Human artificial chromosome (HAC) vector provides long-term therapeutic transgene expression in normal human primary fibroblasts

Introduction of a CD40L genomic fragment via a human artificial chromosome vector permits cell-type-specific gene expression and induces immunoglobulin secretion

“…B cells obtained from CD40L knockout mice were cultured with Jurkat cells carrying mCD40L-HAC (five different clones), intact Jurkat cells, or the stimulated mouse T cells for a week, and then the culture medium was harvested. The concentration of mouse immunoglobulin (Ig)G in the culture medium was measured using enzyme-linked immunosorbent assay (ELISA) and mesenchymal stem cells, providing long-term expression of the therapeutic gene and conferring a growth advantage on cells carrying the vector (Kakeda et al 2005;Ren et al 2005;Yamada et al 2006). In this study, we addressed the use of the HAC vector for treating a genetic disorder caused by a gene deficiency, which requires intrinsic gene expression.…”

“…The mCD40L-HAC vector remaining in the CHO cells was transferred to 1 9 10 7 Jurkat or U937 cells via microcellmediated chromosome transfer (MMCT), as previously described (Doherty and Fisher 2003;Katoh et al 2004;Kakeda et al 2005).…”

Non-Viral Gene Therapy Vectors Carrying Genomic Constructs

“…One approach towards constructing HACs, called "top-down", involves fragmentation of already existing chromosomes and generation of smaller mini-chromosomes, where only the three functional chromosomal elements remain. Several studies have shown that minichromosomes can host and allow the expression of large therapeutic genes, be transferred between various mouse and human cell lines and be transmitted through the mouse germ line (Kakeda et al, 2005;Shen et al, 2001;Voet et al, 2001). Though mini-chromosomes have useful properties for application in transgenics, their use in gene therapy is restricted to an ex vivo approach only.…”

De novo formed satellite DNA-based mammalian artificial chromosomes and their possible applications

“…Recently gene-loaded 21ΔqHACs have been transferred to human primary fibroblasts (Kakeda et al 2005) and to human hematopoietic stem cells (HSCs) (Yamada et al 2006) at clinically relevant frequencies of 1.26 x 10 -4 and 4.0 x 10 -4 , respectively. Nevertheless, despite these increased transfer efficiencies, during the process of microcell formation the host cell generates a heterogeneous population of microcells encapsulating endogenous chromosomes as well as MACs.…”

“…Of note, the remaining p arm of 21ΔqHAC is assumed to encode only a rDNA gene tandem array and pericentromeric heterochromatin and hence should be genetically "neutral." Transgenes that have been efficiently loaded and expressed from these vectors include hypoxanthine guanine phosphoribosyl transferase (HPRT) (Ayabe et al 2005;Grimes et al 2001;Mejía et al, 2001) , enhanced green fluorescent protein (EGFP) (Yamada et al 2006), antibody/cytokine receptor chimera (Kawahara et al 2007), erythropoietin (EPO) (Kakeda et al 2005), DNA-dependent protein kinase catalytic subunit (DNA-PKcs, critical for DNA repair) (Otsuki et al 2005), proinsulin (Suda et al 2006), human dystrophin (Messina et al 2009), CD40L (Yamada et al 2008), the human P53 , telomerase , genes for human growth hormone (HGH), polycystic kidney disease (PKD1) and betaglobin (Basu et al 2005a), CFTR (Auriche et al 2002;Rocchi et al 2010), GCH1 (Ikeno et al 2002), factor IX (Breman et al 2008, NBS1 and VHL genes (Kim et al 2011) and STAT3 (Ikeno et al 2009). Several of these genes were loaded as genomic copies carrying all the necessary control regions for proper wild type gene expression.…”