A chromosomal vector including HAC can be transferred into other desired cell lines by microcell-mediated chromosome transfer (MMCT). For MMCT, donor cells (normally mouse A9 cells and CHO cells) are induced to multinucleate their chromosomes (Fig. 7.3). Micronuclei are then forced through the cell membrane to create microcells by centrifugation in the presence of cytochalasin B which disrupts the cytoskeleton [11]. These microcells can be fused to a recipient cell line under appropriate conditions.

MMCT comprises multiple steps: (1) induction of micronuclei in donor cells, (2) isolation of microcells from micronucleated donor cells, (3) fusion of microcells with recipient cells, (4) integration of the transferred chromosome into host cell nucleus, and (5) selection of microcell hybrid cells by drug selection (Fig. 7.3). Eventual efficiency is determined by the summation of contribution from each step. Established cell lines such as pluripotent stem cells including embryonic stem cells, induced pluripotent stem cells, and somatic stem cells have been preferably used as recipient cells. Efficiency of MMCT has been described by the ratio of drug-resistant colony number to recipient cells. Depending on the type of recipient cells, the efficiency is generally 10⁻⁵–10⁻⁶ when using polyethylene glycol (PEG) for the fusion of microcells and recipient cells. With the advent of stem cell studies, the target of MMCT has been extended to primary cells with finite life span, somatic stem cells, and induced pluripotent stem (iPS) cells. Improvement of the efficiency has emerged as an issue to be resolved.

The microcells conventionally have been fused to recipient cells in the presence of polyethylene glycol (PEG) which acts as a dehydrating agent and fuses plasma membranes. Recently, a more efficient MMCT procedure has been developed [12]. During the course of virus particle formation in the infected cells, viral fusogenic proteins are synthesized de novo, transported to the cell periphery, and presented on the cell surface, followed by extrusion of cell membrane as virus envelope [13]. This well-controlled budding mechanism of envelope virus prompted us to make “a fusogenic microcell” which carries a chromosome to be transmitted and is coated with fusogenic envelope proteins. We chose fusogenic envelope proteins from Measles virus (MV) for which accumulating data have been reported. MV has two envelope glycoproteins, hemagglutinin (H) and fusion (F) proteins, for infection into host cells. Virus particle specifically attaches to the surface of host cells by the interaction between H protein and its receptors on the host cell surface. Binding of the H protein to a receptor triggers the fusion of virus envelope with the host cell membrane by the mediation of F protein. To make “fusogenic microcells,” expression plasmids encoding H and F proteins were transfected into CHO cells carrying a HAC vector. Microcells isolated from the CHO donor cells showed fusion ability to recipient human cells that express a receptor protein CD46, leading to successful transfer of the HAC [12]. It was noted that the MMCT efficiency depended on the expression level of CD46 in recipient cells. CD46 belongs to the family of complement activation regulators that prevent self-cell destruction [14]. Overexpression of CD46 is frequently observed in cancer cells to overcome lysis by complement [15]. Indeed, in the case of fibrosarcoma cell line HT1080 which has high surface density of CD46, the MMCT efficiency was 2 orders of magnitude higher than that with PEG fusion. However, in the case of primary fibroblasts that have low surface density of CD46, the efficiency was comparable to that with PEG fusion. An issue with this method is the narrow range of recipient cells that can be used to obtain high-efficiency fusion. The preceding studies that describe the usage of MV for oncolysis by infection have proposed retargeting of MV by engineering the H protein, i.e., addition of single-chain antibody fragment against surface receptors other than CD46 [16, 17]. The addition of single-chain variable fragment (scFv) against transferrin receptor (TfR) improved fusion efficiency to primary fibroblasts [18]. Although high affinity of scFv to desired surface receptor is not always available with ease, retargeting of MV may be an alternative for PEG-sensitive cells.

Gene therapy has been envisioned to provide a direct and permanent correction of genetic defects. To achieve the desired effects, therapeutic genes need to be carried by safe and effective vectors that can deliver foreign genes to specific cells and thereafter sustain their expression in a physiologically regulated fashion. Gene delivery vectors with the following properties may further add to the applications for autologous gene and cell therapies: (1) high transfer efficiency; (2) long-term stable maintenance in host cells without integration into the host genome; (3) appropriate levels of spatial and temporal expression of therapeutic genes in specifically desired cells; (4) no risk of cellular transformation or stimulation of the host’s immune system; and (5) a system to safeguard against tumor formation. Although a number of different approaches have been attempted to achieve efficient gene transfer and long-term gene expression, this challenging task remains unfulfilled because all current methods have certain limitations, including transient expression, consequent toxicity, undesired immunological response, integration of target genes into the host cell genome, and transcriptional silencing.