1102. Stable, Non-Viral Gene Transfer to Human CD34+ Hematopoietic Progenitor Cells Using the Sleeping Beauty Transposon

Abstract

Primary hematopoietic stem cells (HSC), capable of repopulating the entire hematopoietic and immune system with the full complement of cell types, are an attractive target for gene therapy strategies to treat diseases of blood cells. Gene modification of a patient's HSC could be used for autologous transplantation to treat inherited genetic disorders, including Severe Combined Immune Deficiency (SCID) and other immune deficiencies. Non-viral strategies have become available for gene transfer into cells that result in integration of therapeutic gene cassettes. For example, Sleeping Beauty (SB) transposes directly from one DNA locus to another through the activity of the transposase enzyme recognizing and performing excision and integration of the DNA sequences between specific inverted repeat (IR) sequences of the transposon. Transposase-mediated integration of the IR-flanked expression cassette into chromosomes provides the basis for long-term transgene expression. Here we used the transposon-based gene delivery strategy composed of a two plasmid system: a plasmid containing the SB transposase gene (for transient production of the transposase), and a second plasmid containing an expression cassette with a reporter gene flanked by the SB IR (inverted repeat) sequences. For this study, we evaluated the potential use of the SB transposon system to deliver genes in a stable manner to human hematopoietic cells by electroporation using the Amaxa nucleoporator. One plasmid carrying an enhanced Green Fluorescent Protein (eGFP) reporter cassette (to facilitate FACS analysis of resultant cells) flanked by the SB IR sequences for permanent integration into the target cell chromosomes was co-nucleoporated with a second plasmid carrying the SB transposase expression cassette. We first used the K562 human erythro-leukemia cell line to optimize several parameters of gene delivery, achieving high levels (50-80%) of stable gene integration with optimal amounts and ratios of the two plasmids. Southern analysis of K562 clonal populations showed that 1 to 6 copies of the transposon can be detected. We then nucleoporated the SB-based system into human CD34+ progenitor cells isolated from umbilical cord blood and achieved initial gene transfer to these primary cells at moderate levels (10-30%) that led to stable expression of the reporter gene (1-4%). Results from these studies demonstrate the potential for using the SB transposon system for gene therapy using HSC.