Sleeping Beauty-Mediated Drug Resistance Gene Transfer in Human Hematopoietic Progenitor Cells

Abstract

The Sleeping Beauty (SB) transposon system can insert sequences into mammalian chromosomes, supporting long-term expression of both reporter and therapeutic genes. Hematopoietic progenitor cells (HPCs) are an ideal therapeutic gene transfer target as they are used in therapy for a variety of hematologic and metabolic conditions. As successful SB-mediated gene transfer into human CD34(+) HPCs has been reported by several laboratories, we sought to extend these studies to the introduction of a therapeutic gene conferring resistance to methotrexate (MTX), potentially providing a chemoprotective effect after engraftment. SB-mediated transposition of hematopoietic progenitors, using a transposon encoding an L22Y variant dihydrofolate reductase fused to green fluorescent protein, conferred resistance to methotrexate and dipyridamole, a nucleoside transport inhibitor that tightens MTX selection conditions, as assessed by in vitro hematopoietic colony formation. Transposition of individual transgenes was confirmed by sequence analysis of transposon-chromosome junctions recovered by linear amplification-mediated PCR. These studies demonstrate the potential of SB-mediated transposition of HPCs for expression of drug resistance genes for selective and chemoprotective applications.

Figure 1.

Transposons used in this study. Maps of pKT2/mCAGGs-DHFR(Tyr-22)-eGFP, pKT2/mCAGGs-eGFP, and hyperactive pCMV-SB100x are shown. eGFP, enhanced green fluorescent protein gene; DHFR, dihydrofolate reductase; mCAGGs, mini-CAGGs; pA, poly(A); CMV, cytomegalovirus promoter. Color images available online at www.liebertpub.com/hum

Figure 2.

CD34⁺ cells selectively express GFP 2 or 3 days after nucleofection of a mixed population of CD34-positive and -negative cells. (A) Representative flow cytometric analysis of CD34⁺GFP⁺ cells. Cells were gated on forward scatter (FSC) and side scatter (SSC) and stained with antibodies to CD34 and CD38. (B) Enhanced GFP expression by both CD34⁺CD38^high cells (open column portions) and CD34⁺CD38^low cells (gray column portions) was similar in the presence and absence of SB100x, 2–3 days post nucleofection. A mixture of CD34⁺ and CD34⁻ cells was nucleofected with plasmids encoding SB transposon with or without CMV-SB100x plasmid. DHFR-GFP, pKT2/mCAGGs-DHFR-eGFP; GFP, pKT2/mCAGGs-eGFP. n=4. Color images available online at www.liebertpub.com/hum

Figure 3.

Long-term expression of GFP and methotrexate (MTX) resistance by hematopoietic progenitor colonies after transposition with transposon encoding GFP or DHFR(Tyr-22)-eGFP fusion gene and SB100x. (A) eGFP expression by both types of progenitor colonies after nucleofection of CD34⁺ cells followed by culture in semisolid methylcellulose medium without selection. Open column portions, CFU-GM colonies; solid column portions, erythroid colonies. (B) Half of drug-resistant CFU-GM (51%) and erythroid colonies (56%) were bright enough to be scored GFP⁺ by fluorescence microscopy when plated under selective conditions. Gray column portions, GFP⁺ MTX-resistant colonies; open column portions, GFP⁻ MTX-resistant colonies. CFU-GM, granulocyte and/or monocyte colonies; DG, pKT2/mCAGGs-DHFR-eGFP; GFP, pKT2/mCAGGs-eGFP. n=3 or 4.

Figure 4.

Enhanced GFP expression by drug-resistant hematopoietic progenitor colonies ranged from bright (A) to dim (C) after 12–14 days of drug selection with MTX and dipyridamole. CD34⁺ cells were nucleofected in the presence of pKT2/mCAGGs-DHFR-eGFP transposon (Tn) with and without pCMV-SB100x plasmid as described in MATERIALS AND METHODS.