Prospects for the Use of Artificial Chromosomes and Minichromosome-Like Episomes in Gene Therapy

Abstract

Artificial chromosomes and minichromosome-like episomes are large DNA molecules capable of containing whole genomic loci, and be maintained as nonintegrating, replicating molecules in proliferating human somatic cells. Authentic human artificial chromosomes are very difficult to engineer because of the difficulties associated with centromere structure, so they are not widely used for gene-therapy applications. However, OriP/EBNA1-based episomes, which they lack true centromeres, can be maintained stably in dividing cells as they bind to mitotic chromosomes and segregate into daughter cells. These episomes are more easily engineered than true human artificial chromosomes and can carry entire genes along with all their regulatory sequences. Thus, these constructs may facilitate the long-term persistence and physiological regulation of the expression of therapeutic genes, which is crucial for some gene therapy applications. In particular, they are promising vectors for gene therapy in inherited diseases that are caused by recessive mutations, for example haemophilia A and Friedreich's ataxia. Interestingly, the episome carrying the frataxin gene (deficient in Friedreich's ataxia) has been demonstrated to rescue the susceptibility to oxidative stress which is typical of fibroblasts from Friedreich's ataxia patients. This provides evidence of their potential to treat genetic diseases linked to recessive mutations through gene therapy.

Telomere length homeostasis and telomere position effect on a linear human artificial chromosome are dictated by the genetic background

“…Human artificial chromosomes combine unique properties for transgenic (1) and putative gene therapeutic (2) applications and are essential to study the function and architecture of chromosome structural elements (3–6)—mainly centromeres and telomeres.…”

Delivery of the 135 kb human frataxin genomic DNA locus gives rise to different frataxin isoforms

“…It thus seems that an optimal gene therapy approach for FRDA should guarantee physiologically-regulated expression of the frataxin gene. Indeed, the use of large DNA molecules containing whole genomic loci has recently received attention as an emerging alternative to the use of cDNAs within viral vectors for gene therapy [18,31]. Studies performed in transgenic mice have demonstrated the benefits of using large fragments of genomic DNA, which allow the delivery of intact mammalian genes with all their regulatory elements.…”

“…Amplicon vectors based on herpes simplex virus 1 (HSV-1) are one of the most promising alternatives for the delivery of large DNA molecules in gene therapy protocols as they can accommodate DNA molecules as big as 150 kb [14,31]. This makes them ideal vehicles for delivering entire genomic loci with all their regulatory elements [41,42].…”

Non-Viral Gene Therapy Vectors Carrying Genomic Constructs

“…To achieve this, they must contain the minimal elements required for chromosome function, namely an origin of replication, telomeres and centromeres (Pérez-Luz & Díaz-Nido, 2010). 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.…”

“…The elements responsible for controlled and tissue-specific expression of a gene usually lie within the introns and the sequences before and after the gene. Therefore, the use of genomic constructs which contain the introns and flanking DNA of the therapeutic gene is expected to be more effective than that of minigene constructs in gene therapy for certain genetic diseases where precise levels of the gene product are required (reviewed by (Pérez-Luz & Díaz-Nido, 2010)). Bacterial Artificial Chromosomes (BACs), originating from the human genome project, contain genomic loci of approximately 180 kb on average and cover the entire human genome (Osoegawa et al, 2001).…”