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

Abstract

BackgroundHuman 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.ResultsHere, 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.ConclusionsRetargeted 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0142-z) contains supplementary material, which is available to authorized users.

Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells

“…Other cell types and species, including human, can be employed as recipients in the cell fusion process, widening the variety of tissue types beyond fibroblasts (Hiratsuka et al 2015; Liskovykh et al 2016; Suzuki et al 2016). In addition, selectable markers such as neomycin resistance can be used instead of TK1, alleviating concerns about the perturbation of nucleotide synthesis pathways by HAT medium (Aoki et al 2014).…”

Reversible immortalisation enables genetic correction of human muscle progenitors and engineering of next‐generation human artificial chromosomes for Duchenne muscular dystrophy

“…Overall, we expect the translational journey of HAC‐based cell therapy to be complex, but no more challenging than those of other gene therapy vectors (Tedesco, ). However, some hurdles still need to be overcome, such as the low efficiency of HAC transfer to recipient cells: new technologies to improve this aspect have been recently developed and are currently under investigation in our laboratories (Hiratsuka et al , ; Suzuki et al , ). Lastly, as much as DMD gene therapy relies on size/complexity of the dystrophin copy delivered to dystrophic cells, efficacy of cell therapy for DMD is linked to the amount of stem/progenitor cells able to reach and engraft the dystrophic muscles and their myogenic potency.…”

“…CHO(DYS‐HAC), A9(DYS‐HAC), riDMD(DYS‐HAC2) mesoangioblasts and riDMD(DYS‐HAC2) myoblasts hybrids were generated by microcell‐mediated chromosome transfer (MMCT) as previously described (Koi et al , ; Katoh et al , ; Kazuki et al , ; Hiratsuka et al , ). Detailed protocols can be found in .…”

Monochromosomal Hybrids and Chromosome Transfer: A Functional Approach for Gene Identification

“…It is noteworthy that chromosome transfer technique is being improved for better efficiency and a variety of biomedical applications (69)(70)(71)(72)(73)(74). In the post-genomic era, however, the focus of geneticists and molecular biologists is shifting from genes to proteins, an area termed "proteomics" (75).…”