Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors

Abstract

The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34⁺ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is ∼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34⁺ cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34⁺ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4-8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.

Keywords: chromosomal integration; gene therapy; gene vectors; hematopoietic stem cells; nonviral gene delivery; transposition.

Graphical abstract

Figure 1

Minicircle-Based Sleeping Beauty Transposon Vectors and Cellular Toxicity of Gene Delivery 2 Days Post-nucleofection in CD34⁺ Cells (A) MC vectors encoding the transposon or the transposase component of the SB system are derived from parental plasmids by intramolecular recombination. The parental transposon plasmid carries a CAGGS-Venus expression cassette (green) flanked by the ITRs of SB (purple), plasmid backbone sequences (red), and recombination sites (black). The parental transposase plasmid carries a CMV-SB100X expression cassette (blue), plasmid backbone sequences (red), and recombination sites (black). MCs have a markedly reduced size and lack most of the plasmid backbone sequences. (B) Percentage of DAPI-negative cells determined by flow cytometry. Data are expressed as means ± SEM (n = 5–6 per group). Asterisks indicate significant differences as determined by Student’s t test (*p < 0.05 and **p < 0.01; 10 + 5 or 10 + 10 indicate the μg of the MC-Venus and the SB-transposase used during nucleofection).

Figure 2

Efficiency of Transient Gene Expression following Minicircle-Based Delivery of Sleeping Beauty Transposon Vectors in CD34⁺ Cells (A and B) The (A) percentage and (B) mean fluorescence intensity (MFI) of Venus⁺ cells determined by flow cytometry 2 days post-delivery. (C) Overall efficiency of transient gene delivery and expression calculated by multiplying the percentage of Venus⁺ cells and their MFIs. Data are expressed as means ± SEM (n = 5–6 per group). Asterisks indicate significant differences as determined by Student’s t test (*p < 0.05 and **p < 0.01).

Figure 3

Long-Term Gene Expression following Minicircle-Based Delivery of Sleeping Beauty Transposon Vectors in CD34⁺ Cells (A and B) The (A) percentage and (B) mean fluorescence intensity (MFI) of Venus⁺ cells determined by flow cytometry 15 days post-delivery. (C) Overall efficiency of gene expression calculated by multiplying the percentage of Venus⁺ cells and their MFIs. (D) Relative transposition efficiency determined by the percentage of Venus⁺ cells at day 2 post-nucleofection that retained Venus expression at day 15. Data are expressed as means ± SEM (n = 5–6 per group). Asterisks indicate significant differences as determined by Student’s t test (*p < 0.05 and **p < 0.01).

Figure 4

Stable Gene Transfer and Expression Mediated by Sleeping Beauty Transposons in Committed Hematopoietic Progenitor Cells (A) Representative images of colonies generated after culture of nucleofected cells in methylcellulose medium enriched with a cytokine cocktail supporting the formation of burst-forming unit-erythroid (BFU-E), colony-forming unit-erythroid (CFU-E), colony-forming unit-granulocyte, macrophage (CFU-GM), and colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM). (B) Percentage of colonies stably expressing Venus after MC DNA only and MC DNA/SNIM.RNA-based delivery of the SB transposon system. Data are expressed as means ± SEM (n = 3 per group). Asterisks indicate significant differences as determined by Student’s t test (*p < 0.05 and **p < 0.01).

Figure 5

Vector Copy Numbers in Sleeping Beauty-Engineered CD34⁺ Cells (A) Determination of vector copy number (VCN) in bulk cells cultured for 3 weeks in liquid culture. Data are expressed as means ± SEM (n = 5–6 per group). (B) Correlation between VCN determined in bulk cells and relative stable gene delivery efficiency defined as percentage Venus⁺ cells multiplied with their MFIs. (C) VCN determined in CFUs. r, Pearson coefficient of the correlation; p, significance coefficient.

Figure 6

Genome-wide Distribution of Sleeping Beauty Transposon, MLV-Derived Gammaretroviral, and HIV-1-Derived Lentiviral Integrations in CD34⁺ Cells (A) Distribution of MLV, HIV, and SB insertions in functional genomic segments of human G-CSF-mobilized HSPCs. Numbers show relative enrichment above the random frequency (set to 1). Color intensities depict the degree of deviation from the expected random distribution. The cladogram was drawn on the basis of row means. (B) Distribution of vector insertion sites around transcriptional start sites (TSSs). The gray line corresponds to random insertion frequency. (C) Correlation between integration rates and transcriptional activity of the insertion sites. The numbers of the x axis stand for groups of transcription units of increasing activity in human HSPCs. Negative and positive values of the y axis indicate under- and over-representation in fold change over the random expected insertion frequency, shown as 1. (D) Representation of vector insertion sites cosrresponding to genomic safe harbor criteria. The numbers represent percent values of all insertions of the corresponding group. Color intensities imply deviation from an ideal 100% representation. (E) Overall representation of insertion sites in genomic safe harbors.

Figure 7

Analysis of Engraftment and Gene Expression in NSG Immunodeficient Mice Transplanted with Human CD34⁺ Cells Nucleofected with Different Sleeping Beauty Vector Components (A) Analysis of human hematopoietic engraftment determined by the proportion of human CD45⁺ cells in the bone marrow of NSG mice transplanted with control CD34⁺ cells (Ctrl, not nucleofected, black and white dots or nucleofected without DNA, gray dots), or with CD34⁺ cells nucleofected with two different SB vector component combinations: MC/SB (10 μg Venus MC transposon + 5 μg MC SB transposase) and MC/SNIM.RNA-SB (10 μg Venus MC transposon + 5 μg RNA SB transposase). Black dots correspond to cells maintained for 1–2.5 days in culture before transplantation, whereas white dots correspond to incubations for 4–8 days. Gray dots correspond to nucleofected cells without DNA and incubated for 1–2.5 days. The gray area represents the limit considered for positive engraftment. In all instances, mice were analyzed at 3–4 months post-infusion. (B) Analysis of the percentage of Venus-expressing cells in human hematopoietic cells (CD45⁺) corresponding to animals repopulated with human cells (≥1% CD45⁺ cells in the BM of mice shown in (A). (C) Percentage of Venus⁺ cells in the myeloid (CD33⁺) and lymphoid (CD19⁺) population, as well as in hematopoietic progenitors (CD34⁺) corresponding to animals shown in (B). Data are expressed as mean ± SEM. Asterisks indicate significant differences as determined by Student’s t test (*p < 0.05). Each dot represents data obtained from a single NSG-transplanted recipient. Data from 5 independent experiments are shown.