248. A Bona Fide Mammalian Replicator Enhances All Aspects of Episomal Gene Transfer Into Human Hematopoietic Progenitor Cells

Abstract

Episomal gene transfer for gene therapy applications is limited at present mainly because of luck of long term persistence of episomes in the nucleus of cells. However, episomal vectors may offer a valid alternative to viral vectors as well as to gene editing strategies. To expand the potential of episomal vectors, we used the Initiation of Replication region, (IR), from β-globin gene locus, which is capable to promote DNA replication ectopically (Wang L et al 2004) and can rescue DNA replication within the context of Scaffold /Matrix Attachment Region (S/MAR) based episomal vector (Giannakoppulos A. et al 2009). We constructed two episomal vectors, derived from the protopype S/MAR based episomal vector pEPI-eGFP (6.7Kb), namely, pEP-IR and its control pEPI-SFFV, by replacing CMV promoter of eGFP by the Spleen Focus forming Virus (SFFV) promoter, while vector pEP-IR carries the IR element just upstream the SFFV promoter. Stress Induced Duplex Destabilization (SIDD) analysis of the novel plasmids indicated that these changes would not disrupt their function as episomal vectors and could be beneficial. All three vectors (pEP-IR and control vectors pEPI-SFFV and pEPI-eGFP) showed excellent episomal properties upon transfer into K562 cells. Additionally, they showed transfection efficiency around 30% upon transfer into CD34+ cells from peripheral blood, from adult, mobilised, healthy donors. CD34+/eGFP+ cells sorted by FACS were placed in semisolid cultures and produced differentiated cell-colonies within 14 days. Non-fluorescent colonies do not contain plasmids, as documented previously too (Papapetrou E. et al 2006). Vector pEP-IR shows an enhancement of all aspects of vector performance: 100% fluorescent colonies against (0%) for pEPI-eGFP as found previously (Papapetrou E. et al 2006) and 56% for pEPI-SFFV, in Fluorescent microscopy; increase of plasmid copy number per fluorescent cell, which was 1.78 for pEP-IR against 1.12 for pEPI-SFFV, as estimated by Real Time PCR. The ratio of the number of fluorescent, stably transfected colonies to the number of transfected, seeded cells presenting the rate of establishment in semisolid cultures of transfected CD34+ cells, was 5.5% for pEPI-SFFV and 15% for pEP-IR. It is thus determined that the relative capacity for establishment for pEP-IR is 3-fold higher than that of control vector pEPI-SFFV. Finally, estimation of eGFP mRNA showed that pEP-IR supports 3 times higher eGFP expression per plasmid copy, compared to pEPI-SFFV. Vector pEPI-IR in a series of over five transfections of CD34+ cells, routinely produced fluorescent cells in the area of 99.8%. Practically, vector pEP-IR is expected to be established in all cells that give rise to the differentiated cell-progeny of CD34+ cells, that is in 10% to 20% of the total sorted CD34+/eGFP+ cells. These estimates for vector pEPI-IR can be of clinical importance.