Production of Retroviral Vectors in Primary Human Keratinocytes after DNA-Mediated Gene Transfer Leads to Prolonged Gene Expression

Abstract Amphotropic helper-free retroviral vectors containing either the bacterial neomycin phosphotransferase gene (NEO) or a mutant dihydrofolate reductase gene (DHFR*) were used to infect canine hematopoietic progenitor cells. In previous experiments, successful transfer and expression of both genes in canine CFU-GM were achieved after 24-hour cocultivation with virus-producing cells. The average rate of gene expression was 10% (6% to 16%) as measured by the number of CFU-GM resistant to either the aminoglycoside G418 or methotrexate. In an attempt to increase the efficiency of gene transfer, marrow was cocultured for 24 hours with either NEO or DHFR* virus-producing packaging cells and then kept in long-term marrow culture fed three times with virus-containing supernatant (2 to 5 x 10(6) CFU/mL). After six days, cells were harvested and cultured in CFU-GM assay with and without a selective agent. The average rate of gene expression in CFU- GM in five independent experiments was 46% and ranged from 19% to 87%. In conclusion, the efficiency of gene transfer into canine hematopoietic progenitor cells has been increased fourfold by combining cocultivation with long-term marrow culture as compared with results obtained with cocultivation only.

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Platelet-Released Growth Factors Induce Differentiation of Primary Keratinocytes

Mediators of Inflammation ◽

10.1155/2017/5671615 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 3

Author(s):

Andreas Bayer ◽

Mersedeh Tohidnezhad ◽

Justus Lammel ◽

Sebastian Lippross ◽

Peter Behrendt ◽

...

Keyword(s):

Gene Expression ◽

Growth Factors ◽

Terminal Differentiation ◽

Human Keratinocytes ◽

Keratinocyte Differentiation ◽

Dependent Manner ◽

Primary Human Keratinocytes ◽

Transglutaminase 1 ◽

Keratin 1

Autologous thrombocyte concentrate lysates, for example, platelet-released growth factors, (PRGFs) or their clinically related formulations (e.g., Vivostat PRF®) came recently into the physicians’ focus as they revealed promising effects in regenerative and reparative medicine such as the support of healing of chronic wounds. To elucidate the underlying mechanisms, we analyzed the influence of PRGF and Vivostat PRF on human keratinocyte differentiation in vitro and on epidermal differentiation status of skin wounds in vivo. Therefore, we investigated the expression of early (keratin 1 and keratin 10) and late (transglutaminase-1 and involucrin) differentiation markers. PRGF treatment of primary human keratinocytes decreased keratin 1 and keratin 10 gene expression but induced involucrin and transglutaminase-1 gene expression in an epidermal growth factor receptor- (EGFR-) dependent manner. In concordance with these results, microscopic analyses revealed that PRGF-treated human keratinocytes displayed morphological features typical of keratinocytes undergoing terminal differentiation. In vivo treatment of artificial human wounds with Vivostat PRF revealed a significant induction of involucrin and transglutaminase-1 gene expression. Together, our results indicate that PRGF and Vivostat PRF induce terminal differentiation of primary human keratinocytes. This potential mechanism may contribute to the observed beneficial effects in the treatment of hard-to-heal wounds with autologous thrombocyte concentrate lysates in vivo.

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Human β-Globin Gene Expression after Gene Transfer Using Retroviral Vectors

DNA ◽

10.1089/dna.1987.6.573 ◽

1987 ◽

Vol 6 (6) ◽

pp. 573-582 ◽

Cited By ~ 16

Author(s):

NORMA LERNER ◽

STEVEN BRIGHAM ◽

STEPHEN GOFF ◽

ARTHUR BANK

Keyword(s):

Gene Expression ◽

Gene Transfer ◽

Globin Gene ◽

Retroviral Vectors ◽

Globin Gene Expression

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Regulation of parathyroid hormone-related protein gene expression by epidermal growth factor-family ligands in primary human keratinocytes

Journal of Endocrinology ◽

10.1677/joe.0.1810179 ◽

2004 ◽

Vol 181 (1) ◽

pp. 179-190 ◽

Cited By ~ 19

Author(s):

YM Cho ◽

DA Lewis ◽

PF Koltz ◽

V Richard ◽

TA Gocken ◽

...

Keyword(s):

Gene Expression ◽

Growth Factor ◽

Reporter Gene ◽

Human Keratinocytes ◽

Mrna Levels ◽

Related Protein ◽

Primary Keratinocytes ◽

Primary Human Keratinocytes ◽

Parathyroid Hormone Related Protein ◽

Epidermal Growth

Cultured primary human keratinocytes were the first non-cancer-derived cell type reported to produce the humoral hypercalcemia factor, parathyroid hormone-related protein (PTHrP). Emerging evidence suggests that only a subset of keratinocytes produce high levels of PTHrP in vivo. We found that the PTHrP mRNA content of intact human skin was minimal, whereas transcripts were easily detectable in primary keratinocytes derived from those skin samples. We hypothesized that conditions associated with growth in culture activated PTHrP gene expression in primary keratinocytes. In culture, keratinocytes produce a number of epidermal growth factor (EGF)-like ligands (transforming growth factor-alpha, heparin binding-EGF and amphiregulin) and their receptor, ErbB1. Treatment of keratinocytes with a specific erbB1 inhibitor (PD153035) reduced PTHrP mRNA levels by >80% in rapidly growing keratinocytes. Treatment of keratinocytes with reagents that neutralize amphiregulin reduced PTHrP mRNA levels by approximately 60%. Blockade of erbB1 signaling reduces transcription from the endogenous PTHrP P3-TATA promoter. The Ets transcription factor-binding site, 40 bases upstream of the P3 promoter, is required for baseline expression of PTHrP reporter gene constructs in keratinocytes; in addition, cotransfection of Ets-1 and Ets-2 expression vectors activate the reporter gene constructs. Finally, disruption of both ras and raf signaling reduce reporter gene expression by 80%, suggesting that ErbB1 signaling is mediated by the classic ras/MAP kinase pathway. These findings suggest that acquisition of EGF-like ligand expression has the potential to substantially activate PTHrP gene expression in the epidermis.

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Canine model for gene therapy: inefficient gene expression in dogs reconstituted with autologous marrow infected with retroviral vectors

Blood ◽

10.1182/blood.v71.3.742.bloodjournal713742 ◽

1988 ◽

Vol 71 (3) ◽

pp. 742-747 ◽

Cited By ~ 1

Author(s):

RB Stead ◽

WW Kwok ◽

R Storb ◽

AD Miller

Keyword(s):

Gene Expression ◽

Gene Therapy ◽

Gene Transfer ◽

Transient Gene Expression ◽

Retroviral Vectors ◽

Canine Model ◽

Preclinical Model ◽

Hematopoietic Stem ◽

In Vivo Selection

Successful retroviral gene transfer into murine hematopoietic stem cells indicates the potential for somatic gene therapy in the treatment of certain human hereditary diseases. We developed a canine model to test the applicability of these techniques to a preclinical model of human marrow transplantation. Previously we reported that canine CFU-GM could be infected with retroviral vectors carrying either the gene for a mutant dihydrofolate reductase (DHFR) or neomycin phosphotransferase (NEO). This study reports six lethally irradiated dogs transplanted with autologous marrow cocultivated with retroviral vector-producing cells. This procedure conferred drug resistance to 3% to 13% of the CFU- GM. Three dogs infected with either the NEO or DHFR virus engrafted, but we detected no drug-resistant CFU-GM. Three dogs were given marrow infected with a DHFR virus and received methotrexate (MTX) as in vivo selection; all three had evidence of engraftment. In the surviving dog, we detected 0.03% to 0.1% MTX-resistant CFU-GM at 3 to 5 weeks posttransplant during in vivo selection. These results indicate that we can reconstitute lethally irradiated dogs with autologous marrow exposed to retroviral vectors and suggest that gene transfer into hematopoietic cells is feasible on a large scale. However, the low- level transient gene expression indicates that considerable obstacles remain before human gene therapy can be considered.

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Improvement of Gene Expression and Virus Production in the Use of Retroviral Vectors for Gene Transfer

Vectors as Tools for the Study of Normal and Abnormal Growth and Differentiation ◽

10.1007/978-3-642-74197-5_15 ◽

1989 ◽

pp. 165-173

Author(s):

Petra Artelt ◽

Jörg Bartsch ◽

Hansjörg Hauser ◽

Manfred Wirth

Keyword(s):

Gene Expression ◽

Gene Transfer ◽

Virus Production ◽

Retroviral Vectors

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Canine model for gene therapy: inefficient gene expression in dogs reconstituted with autologous marrow infected with retroviral vectors

Blood ◽

10.1182/blood.v71.3.742.742 ◽

1988 ◽

Vol 71 (3) ◽

pp. 742-747 ◽

Cited By ~ 62

Author(s):

RB Stead ◽

WW Kwok ◽

R Storb ◽

AD Miller

Keyword(s):

Gene Expression ◽

Gene Therapy ◽

Gene Transfer ◽

Transient Gene Expression ◽

Retroviral Vectors ◽

Canine Model ◽

Preclinical Model ◽

Hematopoietic Stem ◽

In Vivo Selection

Abstract Successful retroviral gene transfer into murine hematopoietic stem cells indicates the potential for somatic gene therapy in the treatment of certain human hereditary diseases. We developed a canine model to test the applicability of these techniques to a preclinical model of human marrow transplantation. Previously we reported that canine CFU-GM could be infected with retroviral vectors carrying either the gene for a mutant dihydrofolate reductase (DHFR) or neomycin phosphotransferase (NEO). This study reports six lethally irradiated dogs transplanted with autologous marrow cocultivated with retroviral vector-producing cells. This procedure conferred drug resistance to 3% to 13% of the CFU- GM. Three dogs infected with either the NEO or DHFR virus engrafted, but we detected no drug-resistant CFU-GM. Three dogs were given marrow infected with a DHFR virus and received methotrexate (MTX) as in vivo selection; all three had evidence of engraftment. In the surviving dog, we detected 0.03% to 0.1% MTX-resistant CFU-GM at 3 to 5 weeks posttransplant during in vivo selection. These results indicate that we can reconstitute lethally irradiated dogs with autologous marrow exposed to retroviral vectors and suggest that gene transfer into hematopoietic cells is feasible on a large scale. However, the low- level transient gene expression indicates that considerable obstacles remain before human gene therapy can be considered.

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Peripheral blood lymphocytes as target cells of retroviral vector- mediated gene transfer

Blood ◽

10.1182/blood.v83.7.1988.bloodjournal8371988 ◽

1994 ◽

Vol 83 (7) ◽

pp. 1988-1997 ◽

Cited By ~ 5

Author(s):

F Mavilio ◽

G Ferrari ◽

S Rossini ◽

N Nobili ◽

C Bonini ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Gene Transfer ◽

Cell Lines ◽

Peripheral Blood ◽

Retroviral Vectors ◽

Peripheral Blood Lymphocytes ◽

Cell Populations ◽

Target Cells ◽

Specific Cell

Peripheral blood lymphocytes (PBLs) are key target cells for gene therapy of a number of inherited and acquired blood disorders. We have systematically compared four retroviral vectors, designed according to different strategies, for their efficiency in transfer and expression in human PBLs of the same reporter gene. The receptor gene used in the study codes for the human low-affinity nerve growth factor receptor (LNGFR), and is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transduced gene expression by immunofluorescence, with single cell resolution. Peripheral blood mononuclear cells (PBMCs), as well as human hematopoietic cell lines of myeloid and lymphoid origin, were transduced with the four vectors and analyzed for efficiency of gene transfer, integration and stability of vector proviruses, and LNGFR expression at both RNA and protein level. Fluorescence-activated cell sorter analysis of coexpression of LNGFR and lineage-specific cell surface markers was performed in transduced cell lines, PBLs, and T- cell clones to study gene expression on specific cell subpopulations. Although crucial differences were observed among different constructs, all retroviral vectors could transduce, under appropriate infection conditions, T-cell populations representative of the normal immune repertoire. Gene transfer and expression could be demonstrated also in circulating progenitors of mature T cells. Expression of the transduced gene was heterogeneous among cell populations infected with the different vectors, with optimal results obtained by two of the four constructs. Finally, we have devised a simple protocol based on vector- mediated gene transfer and positive immunoselection of the transduced cells that produces virtually 100% gene-modified cells. This may represent a crucial improvement in the way of designing efficacious protocols involving the use of gene-modified T lymphocytes in clinical studies.

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γ-Propoxy-Sulfo-Lichenan Induces In Vitro Cell Differentiation of Human Keratinocytes

Molecules ◽

10.3390/molecules24030574 ◽

2019 ◽

Vol 24 (3) ◽

pp. 574

Author(s):

Stefan Esch ◽

Maren Gottesmann ◽

Andreas Hensel

Keyword(s):

Gene Expression ◽

Wound Healing ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Gene Array ◽

Human Keratinocytes ◽

Keratinocyte Differentiation ◽

Cell Physiology ◽

Dependent Manner ◽

Primary Human Keratinocytes

Background: As non-cellulosic β-d-glucans are known to exert wound-healing activity by triggering keratinocytes into cellular differentiation, the functionality of a semisynthetic lichenan-based polysaccharide on skin cell physiology was investigated. Methods: γ-Propoxy-sulfo-lichenan (γ-PSL, molecular weight 52 kDa, β-1,3/1,4-p-d-Glucose, degree of substitution 0.7) was prepared from lichenan. Differentiation of primary human keratinocytes was assayed by the protein analysis of differentiation specific markers and by gene expression analysis (qPCR). The gene array gave insight into the cell signaling induced by the polysaccharide. Results: γ-PSL (1 to 100 μg/mL) triggered keratinocytes, in a concentration-dependent manner, into the terminal differentiation, as shown by the increased protein expression of cytokeratin 1 (KRT1). Time-dependent gene expression analysis proved differentiation-inducing effects, indicating strong and fast KRT1 gene expression, while KRT10 expression showed a maximum after 12 to 24 h, followed by downregulation to the basal level. Involucrin gene expression was only changed to a minor extent, which was similar to loricrin and transglutaminase. Gene array indicated the influence of γ-PSL on MAP kinase and TGF-β mediated signaling towards keratinocyte differentiation. Conclusion: The propoxylated lichenan may improve wound healing by topical application to promote the terminal barrier formation of keratinocytes.

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Peripheral blood lymphocytes as target cells of retroviral vector- mediated gene transfer

Blood ◽

10.1182/blood.v83.7.1988.1988 ◽

1994 ◽

Vol 83 (7) ◽

pp. 1988-1997 ◽

Cited By ~ 179

Author(s):

F Mavilio ◽

G Ferrari ◽

S Rossini ◽

N Nobili ◽

C Bonini ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Gene Transfer ◽

Cell Lines ◽

Peripheral Blood ◽

Retroviral Vectors ◽

Peripheral Blood Lymphocytes ◽

Cell Populations ◽

Target Cells ◽

Specific Cell

Abstract Peripheral blood lymphocytes (PBLs) are key target cells for gene therapy of a number of inherited and acquired blood disorders. We have systematically compared four retroviral vectors, designed according to different strategies, for their efficiency in transfer and expression in human PBLs of the same reporter gene. The receptor gene used in the study codes for the human low-affinity nerve growth factor receptor (LNGFR), and is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transduced gene expression by immunofluorescence, with single cell resolution. Peripheral blood mononuclear cells (PBMCs), as well as human hematopoietic cell lines of myeloid and lymphoid origin, were transduced with the four vectors and analyzed for efficiency of gene transfer, integration and stability of vector proviruses, and LNGFR expression at both RNA and protein level. Fluorescence-activated cell sorter analysis of coexpression of LNGFR and lineage-specific cell surface markers was performed in transduced cell lines, PBLs, and T- cell clones to study gene expression on specific cell subpopulations. Although crucial differences were observed among different constructs, all retroviral vectors could transduce, under appropriate infection conditions, T-cell populations representative of the normal immune repertoire. Gene transfer and expression could be demonstrated also in circulating progenitors of mature T cells. Expression of the transduced gene was heterogeneous among cell populations infected with the different vectors, with optimal results obtained by two of the four constructs. Finally, we have devised a simple protocol based on vector- mediated gene transfer and positive immunoselection of the transduced cells that produces virtually 100% gene-modified cells. This may represent a crucial improvement in the way of designing efficacious protocols involving the use of gene-modified T lymphocytes in clinical studies.

Download Full-text