Limits...
Retargeting of microcell fusion towards recipient cell-oriented transfer of human artificial chromosome.

Hiratsuka M, Ueda K, Uno N, Uno K, Fukuhara S, Kurosaki H, Takehara S, Osaki M, Kazuki Y, Kurosawa Y, Nakamura T, Katoh M, Oshimura M - BMC Biotechnol. (2015)

Bottom Line: Human artificial chromosome (HAC) vectors have some unique characteristics as compared with conventional vectors, carrying large transgenes without size limitation, showing persistent expression of transgenes, and existing independently from host genome in cells.Retargeted MV-MMCT using chimeric H protein with scFvs succeeded in extending the cell spectrum for gene transfer via HAC vectors.Therefore, this technology could facilitate the systematic cell engineering by HACs.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cell Genetics, Department of Molecular and Cellular Biology, School of Life Sciences, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503, Japan. mhiratsu@med.tottori-u.ac.jp.

ABSTRACT

Background: Human artificial chromosome (HAC) vectors have some unique characteristics as compared with conventional vectors, carrying large transgenes without size limitation, showing persistent expression of transgenes, and existing independently from host genome in cells. With these features, HACs are expected to be promising vectors for modifications of a variety of cell types. However, the method of introduction of HACs into target cells is confined to microcell-mediated chromosome transfer (MMCT), which is less efficient than other methods of vector introduction. Application of Measles Virus (MV) fusogenic proteins to MMCT instead of polyethylene glycol (PEG) has partly solved this drawback, whereas the tropism of MV fusogenic proteins is restricted to human CD46- or SLAM-positive cells.

Results: Here, we show that retargeting of microcell fusion by adding anti-Transferrin receptor (TfR) single chain antibodies (scFvs) to the extracellular C-terminus of the MV-H protein improves the efficiency of MV-MMCT to human fibroblasts which originally barely express both native MV receptors, and are therefore resistant to MV-MMCT. Efficacy of chimeric fusogenic proteins was evaluated by the evidence that the HAC, tagged with a drug-resistant gene and an EGFP gene, was transferred from CHO donor cells into human fibroblasts. Furthermore, it was demonstrated that no perturbation of either the HAC status or the functions of transgenes was observed on account of retargeted MV-MMCT when another HAC carrying four reprogramming factors (iHAC) was transferred into human fibroblasts.

Conclusions: Retargeted MV-MMCT using chimeric H protein with scFvs succeeded in extending the cell spectrum for gene transfer via HAC vectors. Therefore, this technology could facilitate the systematic cell engineering by HACs.

No MeSH data available.


Membrane-fusion activity of Haals-αTfR in recipient cells. (A) Detection of surface expression of target receptors in recipient cells. Surface expression of TfR in HT1080 and HFL-1 cells was analyzed with flow cytometry by staining with PE-conjugated anti-TfR antibody (black peak) or an isotype control (white peak). (B) Schematic representation of recombinant H protein. scFv is displayed as a C-terminal extension of H glycoprotein. N; Amino-terminal cytoplasmic tail, TM; Transmembrane domain, *; Y481A, R533A, S548L and F549S mutations in H protein. (C) Syncytium formation ability differed among scFv clones. HT1080 cells were co-transfected with F and indicated H expression plasmid, and were photographed 30 hr later. (D) Fusion test by co-culture assay of donor and recipient cells. CHO cells stably expressing the F and H proteins were co-cultured with HFL-1, and were photographed 24 hr later. Yellow arrows indicate syncytia. (E) The number of resistant/GFP(+) colonies from HFL-1 cells by MV-MMCT using H or Haals-TfR. Data are the means of four independent experiments (±SD), **; p < 0.01 (unpaired t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4472177&req=5

Fig1: Membrane-fusion activity of Haals-αTfR in recipient cells. (A) Detection of surface expression of target receptors in recipient cells. Surface expression of TfR in HT1080 and HFL-1 cells was analyzed with flow cytometry by staining with PE-conjugated anti-TfR antibody (black peak) or an isotype control (white peak). (B) Schematic representation of recombinant H protein. scFv is displayed as a C-terminal extension of H glycoprotein. N; Amino-terminal cytoplasmic tail, TM; Transmembrane domain, *; Y481A, R533A, S548L and F549S mutations in H protein. (C) Syncytium formation ability differed among scFv clones. HT1080 cells were co-transfected with F and indicated H expression plasmid, and were photographed 30 hr later. (D) Fusion test by co-culture assay of donor and recipient cells. CHO cells stably expressing the F and H proteins were co-cultured with HFL-1, and were photographed 24 hr later. Yellow arrows indicate syncytia. (E) The number of resistant/GFP(+) colonies from HFL-1 cells by MV-MMCT using H or Haals-TfR. Data are the means of four independent experiments (±SD), **; p < 0.01 (unpaired t-test).

Mentions: To explore applicability of MV-MMCT to human fibroblasts, TfR was selected as the target receptor in giving a new directivity for MV-H protein, because TfR is known to be ubiquitously expressed in all tissues [31]. Flow cytometric analysis demonstrated that TfR was highly expressed in both HFL-1 and HT1080 cells (Figure 1A), whereas CD46 was expressed only in HT1080 cells, and very rarely in HFL-1 cells [19]. Subsequently, recombinant retargeted H proteins were constructed by using 8 clones of anti-TfR scFvs (Additional file 1 Table S1), which recognized different epitopes of TfR, from the phage-display antibody library [32]. Anti-TfR scFvs were fused to the C terminus of a quadruple mutated H protein (Haals: Y481A, R533A, S548L and F549S), which lacked the ability to bind both CD46 and SLAM [27,33], to validate the effect of anti-TfR scFvs on cell fusion more precisely (Figure 1B). To screen 8 constructs of chimeric H proteins (Haals-αTfRs), we transfected expression plasmids encoding MV-F and Haals-αTfRs into HT1080 cells and assayed syncytium formation by homofusion. Sufficient formation of syncytia and syncytium-induced cell death was detected in clone No.1, No.5 and No.6, whereas a very low number of syncytia were formed in other clones (Figure 1C). Thus, clone No.5 (Haals-αTfR-5) was selected for use in further experiments.Figure 1


Retargeting of microcell fusion towards recipient cell-oriented transfer of human artificial chromosome.

Hiratsuka M, Ueda K, Uno N, Uno K, Fukuhara S, Kurosaki H, Takehara S, Osaki M, Kazuki Y, Kurosawa Y, Nakamura T, Katoh M, Oshimura M - BMC Biotechnol. (2015)

Membrane-fusion activity of Haals-αTfR in recipient cells. (A) Detection of surface expression of target receptors in recipient cells. Surface expression of TfR in HT1080 and HFL-1 cells was analyzed with flow cytometry by staining with PE-conjugated anti-TfR antibody (black peak) or an isotype control (white peak). (B) Schematic representation of recombinant H protein. scFv is displayed as a C-terminal extension of H glycoprotein. N; Amino-terminal cytoplasmic tail, TM; Transmembrane domain, *; Y481A, R533A, S548L and F549S mutations in H protein. (C) Syncytium formation ability differed among scFv clones. HT1080 cells were co-transfected with F and indicated H expression plasmid, and were photographed 30 hr later. (D) Fusion test by co-culture assay of donor and recipient cells. CHO cells stably expressing the F and H proteins were co-cultured with HFL-1, and were photographed 24 hr later. Yellow arrows indicate syncytia. (E) The number of resistant/GFP(+) colonies from HFL-1 cells by MV-MMCT using H or Haals-TfR. Data are the means of four independent experiments (±SD), **; p < 0.01 (unpaired t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4472177&req=5

Fig1: Membrane-fusion activity of Haals-αTfR in recipient cells. (A) Detection of surface expression of target receptors in recipient cells. Surface expression of TfR in HT1080 and HFL-1 cells was analyzed with flow cytometry by staining with PE-conjugated anti-TfR antibody (black peak) or an isotype control (white peak). (B) Schematic representation of recombinant H protein. scFv is displayed as a C-terminal extension of H glycoprotein. N; Amino-terminal cytoplasmic tail, TM; Transmembrane domain, *; Y481A, R533A, S548L and F549S mutations in H protein. (C) Syncytium formation ability differed among scFv clones. HT1080 cells were co-transfected with F and indicated H expression plasmid, and were photographed 30 hr later. (D) Fusion test by co-culture assay of donor and recipient cells. CHO cells stably expressing the F and H proteins were co-cultured with HFL-1, and were photographed 24 hr later. Yellow arrows indicate syncytia. (E) The number of resistant/GFP(+) colonies from HFL-1 cells by MV-MMCT using H or Haals-TfR. Data are the means of four independent experiments (±SD), **; p < 0.01 (unpaired t-test).
Mentions: To explore applicability of MV-MMCT to human fibroblasts, TfR was selected as the target receptor in giving a new directivity for MV-H protein, because TfR is known to be ubiquitously expressed in all tissues [31]. Flow cytometric analysis demonstrated that TfR was highly expressed in both HFL-1 and HT1080 cells (Figure 1A), whereas CD46 was expressed only in HT1080 cells, and very rarely in HFL-1 cells [19]. Subsequently, recombinant retargeted H proteins were constructed by using 8 clones of anti-TfR scFvs (Additional file 1 Table S1), which recognized different epitopes of TfR, from the phage-display antibody library [32]. Anti-TfR scFvs were fused to the C terminus of a quadruple mutated H protein (Haals: Y481A, R533A, S548L and F549S), which lacked the ability to bind both CD46 and SLAM [27,33], to validate the effect of anti-TfR scFvs on cell fusion more precisely (Figure 1B). To screen 8 constructs of chimeric H proteins (Haals-αTfRs), we transfected expression plasmids encoding MV-F and Haals-αTfRs into HT1080 cells and assayed syncytium formation by homofusion. Sufficient formation of syncytia and syncytium-induced cell death was detected in clone No.1, No.5 and No.6, whereas a very low number of syncytia were formed in other clones (Figure 1C). Thus, clone No.5 (Haals-αTfR-5) was selected for use in further experiments.Figure 1

Bottom Line: Human artificial chromosome (HAC) vectors have some unique characteristics as compared with conventional vectors, carrying large transgenes without size limitation, showing persistent expression of transgenes, and existing independently from host genome in cells.Retargeted MV-MMCT using chimeric H protein with scFvs succeeded in extending the cell spectrum for gene transfer via HAC vectors.Therefore, this technology could facilitate the systematic cell engineering by HACs.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cell Genetics, Department of Molecular and Cellular Biology, School of Life Sciences, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503, Japan. mhiratsu@med.tottori-u.ac.jp.

ABSTRACT

Background: Human artificial chromosome (HAC) vectors have some unique characteristics as compared with conventional vectors, carrying large transgenes without size limitation, showing persistent expression of transgenes, and existing independently from host genome in cells. With these features, HACs are expected to be promising vectors for modifications of a variety of cell types. However, the method of introduction of HACs into target cells is confined to microcell-mediated chromosome transfer (MMCT), which is less efficient than other methods of vector introduction. Application of Measles Virus (MV) fusogenic proteins to MMCT instead of polyethylene glycol (PEG) has partly solved this drawback, whereas the tropism of MV fusogenic proteins is restricted to human CD46- or SLAM-positive cells.

Results: Here, we show that retargeting of microcell fusion by adding anti-Transferrin receptor (TfR) single chain antibodies (scFvs) to the extracellular C-terminus of the MV-H protein improves the efficiency of MV-MMCT to human fibroblasts which originally barely express both native MV receptors, and are therefore resistant to MV-MMCT. Efficacy of chimeric fusogenic proteins was evaluated by the evidence that the HAC, tagged with a drug-resistant gene and an EGFP gene, was transferred from CHO donor cells into human fibroblasts. Furthermore, it was demonstrated that no perturbation of either the HAC status or the functions of transgenes was observed on account of retargeted MV-MMCT when another HAC carrying four reprogramming factors (iHAC) was transferred into human fibroblasts.

Conclusions: Retargeted MV-MMCT using chimeric H protein with scFvs succeeded in extending the cell spectrum for gene transfer via HAC vectors. Therefore, this technology could facilitate the systematic cell engineering by HACs.

No MeSH data available.