Signed outside: a surface marker system for transgenic cytoplasmic proteins

Wohlgensinger, Vital; Seger, Reinhard; Ryan, Martin; Reichenbach, Janine; Siler, Ulrich

doi:10.1038/gt.2010.73

Cited by 4 publications

(8 citation statements)

References 21 publications

(16 reference statements)

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“…Lethally irradiated p47 phox-/-mice were reconstituted with Lin-cells transduced with a gammaretroviral vector encoding the DLNGFR-2A-p47 phox fusion construct under miR223 control, allowing for indirect p47 phox detection by DLNGFR surface staining (Wohlgensinger et al, 2010). Significant DLNGFR expression was observed in F4/80+ peritoneal macrophages in all treated animals, indicating miR223 promoter activity in macrophages (Fig.…”

Section: The Mir223 Promoter Drives Myelo-specific Transgene Expressimentioning

confidence: 99%

“…We tested the miR223 promoter in conjunction with the gp91 phox and the p47 phox subunits of the phagocytic NADPH oxidase as well as with a DLNGFR-2A-p47 phox fusion construct (Wohlgensinger et al, 2010) (Supplementary Fig. S1).…”

Section: The Mir223 Promoter Induces Transgene Expression Upon Granulmentioning

confidence: 99%

“…S1; Supplementary Material available online at www.liebertonline.com/hgtb). The gammaretroviral vector encoding SFFV:DLNGFR-2A-p47 phox was already described (Wohlgensinger et al, 2010). In this construct, the SFFV promoter was exchanged against the miR223 promoter.…”

Section: Vector Constructionmentioning

confidence: 99%

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Human miR223 Promoter as a Novel Myelo-Specific Promoter for Chronic Granulomatous Disease Gene Therapy

Brendel

Haenseler

Wohlgensinger

et al. 2013

Human Gene Therapy Methods

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Targeting transgene expression to specific hematopoietic cell lineages could contribute to the safety of retroviral vectors in gene therapeutic applications. Chronic granulomatous disease (CGD), a defect of phagocytic cells, can be managed by gene therapy, using retroviral vectors with targeted expression to myeloid cells. In this context, we analyzed the myelospecificity of the human miR223 promoter, which is known to be strongly upregulated during myeloid differentiation, to drive myeloid-restricted expression of p47 phox and gp91 phox in mouse models of CGD and in primary patient-derived cells. The miR223 promoter restricted the expression of p47 phox , gp91 phox , and green fluorescent protein (GFP) within self-inactivating (SIN) gamma-and lentiviral vectors to granulocytes and macrophages, with only marginal expression in lymphocytes or hematopoietic stem and progenitor cells. Furthermore, gene transfer into primary CD34+ cells derived from a p47 phox patient followed by ex vivo differentiation to neutrophils resulted in restoration of Escherichia coli killing activity by miR223 promoter-mediated p47 phox expression. These results indicate that the miR223 promoter as an internal promoter within SIN gene therapy vectors is able to efficiently correct the CGD phenotype with negligible activity in hematopoietic progenitors, thereby limiting the risk of insertional oncogenesis and development of clonal dominance.

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Section: The Mir223 Promoter Drives Myelo-specific Transgene Expressimentioning

confidence: 99%

Section: The Mir223 Promoter Induces Transgene Expression Upon Granulmentioning

confidence: 99%

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Human miR223 Promoter as a Novel Myelo-Specific Promoter for Chronic Granulomatous Disease Gene Therapy

Brendel

Haenseler

Wohlgensinger

et al. 2013

Human Gene Therapy Methods

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“…The NGFR open reading frame without the RSP (NoRSP-ΔNGFR) was PCR amplified from the plasmid ΔNGFR-2A-p47 (ref. 33 ), as a EcoRI-NotI fragment and inserted in the RSP-MCS-plasmid, in the same open reading frame as the RSP. The cohesin open reading frame was amplified as a XhoI-XhoI fragment and inserted into the RSP-ΔNGFR-MCS-plasmid, between RSP and ΔNGFR in the same open reading frame, producing the RSP-cohesin-NGFR-MCS-plasmid.…”

Section: Methodsmentioning

confidence: 99%

Transcytosis in the blood–cerebrospinal fluid barrier of the mouse brain with an engineered receptor/ligand system

Méndez-Gómez

Galera‐Prat

Meyers

et al. 2015

Molecular Therapy - Methods & Clinical Development

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Crossing the blood–brain and the blood–cerebrospinal fluid barriers (BCSFB) is one of the fundamental challenges in the development of new therapeutic molecules for brain disorders because these barriers prevent entry of most drugs from the blood into the brain. However, some large molecules, like the protein transferrin, cross these barriers using a specific receptor that transports them into the brain. Based on this mechanism, we engineered a receptor/ligand system to overcome the brain barriers by combining the human transferrin receptor with the cohesin domain from Clostridium thermocellum, and we tested the hybrid receptor in the choroid plexus of the mouse brain with a dockerin ligand. By expressing our receptor in choroidal ependymocytes, which are part of the BCSFB, we found that our systemically administrated ligand was able to bind to the receptor and accumulate in ependymocytes, where some of the ligand was transported from the blood side to the brain side.

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“…Transgene expression was driven either by the ubiquitously active spleen focus-forming virus (SFFV) promoter or by the myelospecific microRNA 223 (mir223) promoter 18 . The expression cassette was composed of truncated Low-affinity nerve growth factor receptor (ΔLNGFR) and p47 phox linked by the 2 A self-cleaving peptide 19 . Transduction efficiency reached 8.9% and 11.6% for mir223, and SFFV-driven γ-retroviral vectors ( Fig.…”

mentioning

confidence: 99%

CRISPR/Cas9-generated p47phox-deficient cell line for Chronic Granulomatous Disease gene therapy vector development

Wrona

Siler

Reichenbach

2017

Sci Rep

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Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47phox-deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (ΔGT) mutation in p47phox encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47phox-subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47phox CGD patients carries ΔGT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ΔGT cell line reflects the most frequent form of p47phox-deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.

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Signed outside: a surface marker system for transgenic cytoplasmic proteins

Cited by 4 publications

References 21 publications

Human miR223 Promoter as a Novel Myelo-Specific Promoter for Chronic Granulomatous Disease Gene Therapy

Human miR223 Promoter as a Novel Myelo-Specific Promoter for Chronic Granulomatous Disease Gene Therapy

Transcytosis in the blood–cerebrospinal fluid barrier of the mouse brain with an engineered receptor/ligand system

CRISPR/Cas9-generated p47phox-deficient cell line for Chronic Granulomatous Disease gene therapy vector development

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