Site-specific, Rep-mediated integration of the intact β-globin locus in the human erythroleukaemic cell line K562

Abstract

The stable, regulated and tissue-specific expression of a therapeutic transgene can be best achieved by the transfer of a complete genomic locus, which will include the shortand long-range regulatory elements that are critical for the accurate control of gene expression. However, when techniques that rely on the random integration of exogenous DNA into the human genome are used for gene transfer, the risk of insertional mutagenesis remains a major issue. Using components derived from the adeno-associated virus (AAV), we have successfully targeted the integration of 200 kb bacterial artificial chromosomes containing the entire b-globin locus into the AAVS1 site on human chromosome 19. We show that transient expression of the AAV Rep proteins in K562 cells facilitated site-specific transgene integration in 17% (6 of 36) of all analysed integration sites. Southern blot analysis revealed the locus had integrated into AAVS1 as an intact, functional unit in five of the six clones generated. Furthermore, each of the site-specific integrants exhibited sustained and appropriately regulated transgene gene expression over a period of 8 months of continuous culture in the absence of selective pressure. We anticipate that the approach developed in this study may be suitable for facilitating targeted integration of intact genomic loci in adult and embryonic stem cells, and therefore provide a powerful tool not just for functional studies but in establishing model systems for the ex vivo correction of genetic disorders.

Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin

“…K562 cell lines are often aneuploid 21,22 , and karyotyping of chromosome 11 revealed our lines to be triploid for the b-globin locus ( Supplementary Fig. 5a).…”

Gene Therapy for Hemoglobinopathies

“…In 2008, Howden and colleagues used bacterial artificial chromosomes and components from adeno-associated virus to preferentially insert β-globin into the AAVS1 in K562 cells. 137 Of the 36 insertion sites analyzed, only six of them (17%) occurred in AAVS1, and five out of those six were intact and functional.…”

New Vectors for Stable and Safe Gene Modification

“…Co-transfection of both plasmids in 293T cells achieved site-specific integration in the AAVS1 integration site. This system has achieved stable expression in several cell lines including hematopoietic cells (Howden et al, 2008). However the low plasmid transfection efficiency in primary cells has limited its use in gene therapy applications.…”