Integration-free and stable expression of FVIII using a human artificial chromosome

Abstract

Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts. To examine the copy number effect on the gene expression levels and its stability for a long-term culture for a future application in gene therapy, we constructed a HAC vector carrying the human factor VIII (FVIII) complementary DNA, FVIII-HAC in Chinese hamster ovary (CHO) cells. One and more copies of FVIII gene on the HAC were expressed in the copy-number-dependent manner in the CHO cells. The HAC with 16 copies of FVIII, FVIII (16)-HAC, was transferred from CHO hybrids into a human immortalized mesenchymal stem cell using microcell-mediated chromosome transfer. The expression levels of HAC-derived FVIII transgene products were compared with transfected FVIII plasmids. The former showed expression levels consistent with those of the original clones, even after 50 population doublings, whereas the latter showed a remarkable decrease in expression despite unvarying DNA content, indicating that the gene on the HAC is resistant to gene silencing. These results suggest that the HAC-mediated therapeutic gene-expression system may be a powerful tool for stable expression of transgenes, and possibly for industrial production of gene products.

Bi-HAC Vector System toward Gene and Cell Therapy

“…1b). For gene therapy, a gene of interest can be loaded into the 21HAC vector 22,32,33 while Yamanaka 4 transcription factors (Oct3/4, Sox2, Klf4 and c-Myc) cassette 29 or direct-reprogramming factors 34-36 can be loaded into the tet-O HAC vector that will be eliminated after cell reprogramming. Thus, such bi-HAC-based expression system could be a powerful tool for gene and cell therapy.…”

Human artificial chromosomes for Duchenne muscular dystrophy and beyond: challenges and hopes

“…The deep knowledge on haematopoietic stem cell biology and transplantation would be a major plus for ex vivo gene therapy of this subset of disorders. Finally, the space on the HAC could also be exploited to accommodate more than one gene, different gene functions or several copies of the same gene, as was shown for factor VIII deficiency and haemophilia A (Kurosaki et al 2011).…”

3.2 Chromosome Rearrangements and Gene Amplification

“…The artificial chromosome expression system was developed based on preengineered artificial chromosomes with multiple recombination acceptor sites to improve CHO cell line generation [44][45][46][47][48]. A similar artificial chromosome system using human artificial chromosomes was also used in human factor VIII production in CHO cells [49].…”