1037. Site-Specific Genomic Integration in Hematopoietic Cells

Abstract

Methods of gene transfer to hematopoietic stem cells (HSCs) that result in safe stable chromosomal integration and persistent gene expression in vivo are desired. HSC are an attractive target as their modification could provide treatments for many inherited blood diseases such as Severe Combined Immunodeficiency (SCID) and acquired diseases such as infection by Human Immunodeficiency Virus (HIV-1). PhiC31 integrase is a site-specific recombinase that catalyses a specific, unidirectional integration reaction between two distinct DNA sites known as attB and attP. It has been demonstrated previously that a plasmid expressing the integrase can mediate the integration of a co-delivered attB-containing plasmid into mammalian chromosomes at pseudo attP-sites (host sites sharing homology to attP, as recognized by phiC31). Here we delivered the non-viral phiC31 integrase system to achieve site-restricted integration of genes into the genome of hematopoietic cells using the AMAXA nucleoporator. We applied the phiC31system to integrate a MND-driven eGFP reporter gene into the genomes of human hematopoietic cell lines and primary CD34+ progenitor cells. In the K562 cell line, we observed stable expression of eGFP in >60% of cells for over 2 months; in contrast, in control cells not treated with integrase, the level of gene expression had fallen to near background (<1%) by 2 weeks. In purified human cord blood and bone marrow CD34+ progenitor cells, the integrase repeatedly improved marking efficiency when compared to controls. However, to date relatively low levels of cells that express eGFP (1% range) have been observed over 4-6 weeks of long-term culture. A LAM-PCR based method was applied to investigate pseudo attP-sites in the marked hematopoietic cells. Analyses of integration sites in these marked cells indicated that the preferred pseudo attP-sites chosen by the phiC31 integrase in hematopoietic cells may differ from those previously reported in other commonly used human cell lines and primary cells. Integration events into these pseudo attP-sites are directly responsible for the high level of marking seen in these hematopoietic studies. We are currently defining the identity and expression context afforded by hematopoietic-preferred integration sites. These encouraging results of long-term stable expression highlight the potential of this likely safer non-viral method for ameliorating diseases affecting the hematopoietic system.