Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element

Abstract

Episomally maintained self-replicating systems present attractive alternative vehicles for gene therapy applications. Recent insights into the ability of chromosomal scaffold/ matrix attachment regions (S/MARs) to mediate episomal maintenance of genetic elements allowed the development of a small circular episomal vector that functions independently of virally encoded proteins. In this study, we investigated the potential of this vector, pEPI-eGFP, to mediate gene transfer in hematopoietic progenitor cell lines and primary human cells. pEPI-eGFP was episomally maintained and conferred sustained eGFP expression even in nonselective conditions in the human cell line, K562, as well as in primary human fibroblast-like cells. In contrast, in the murine erythroleukemia cell line, MEL, transgene expression was silenced through histone deacetylation, despite the vector's episomal persistence. Hematopoietic semisolid cell colonies derived from transfected human cord blood CD34 + cells expressed eGFP, albeit at low levels. After 4 weeks, the vector is retained in approximately 1% of progeny cells. Our results provide the first evidence that S/MAR-based plasmids can function as stable episomes in primary human cells, supporting long-term transgene expression. However, they do not display universal behavior in all cell types.

Direct comparison of hepatocyte-specific expression cassettes following adenoviral and nonviral hydrodynamic gene transfer

“…Minicircles containing an SV40 origin of replication and an SMAR were not episomally replicative following partial hepatectomy in the current study. These data are in line with observations of Papapetrou et al 45 who showed that 4 weeks after transduction of human hematopoietic progenitor cells with a plasmid containing an SMAR, DNA was retained in less than 1% of the transduced cells. In contrast, mitotic stability of an episomal vector containing a human SMAR has been shown in in vitro studies.…”

Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector

“…23 Importantly, these properties are conferred without the need for virus-encoded proteins or selective pressure. 21,24 Mitotic stability appears to be provided by direct interaction of the S/MAR element with components of the nuclear matrix, 23,25 including the scaffold attachment factor A protein, which is the principal component of cellular chromatin and chromosomes. 26 Furthermore, this interaction probably brings the S/MAR plasmids into contact with the host replication machinery, which is located on the nuclear matrix, thereby facilitating replication once per cell cycle and hence extra-chromosomal stability.…”

Non-viral S/MAR vectors replicate episomally in vivo when provided with a selective advantage

“…19 The initial selection step, however, is critical to enrich the mitotically stable episomal plasmids in vitro, and lack of selective pressure results in less than 1% of replicating cells retaining the vector after 1 month. 7,20 Both in vitro and in vivo the stable attachment of S/MAR plasmid to the chromatin and its subsequent replication is a very rare event. S/MAR-plasmid attachment appears to require a reorganization of chromatin during cell division.…”