Efficient polymer nanoparticle-mediated delivery of gene editing reagents into human hematopoietic stem and progenitor cells

Abstract

Clinical applications of hematopoietic stem cell (HSC) gene editing are limited due to their complex and expensive logistics. HSC editing is commonly performed ex vivo using electroporation and requires good manufacturing practice (GMP) facilities, similar to bone marrow transplant centers. In vivo gene editing could overcome this limitation; however, electroporation is unsuitable for systemic in vivo applications to HSCs. Here we evaluated polymer-based nanoparticles (NPs), which could also be used for in vivo administration, for the delivery of mRNA and nucleases to human granulocyte colony-stimulating factor (GCSF)-mobilized CD34⁺ cells. NP-mediated ex vivo delivery showed no toxicity, and the efficiency was directly correlated with the charge of the NPs. In a side-by-side comparison with electroporation, NP-mediated gene editing allowed for a 3-fold reduction in the amount of reagents, with similar efficiency. Furthermore, we observed enhanced engraftment potential of human HSCs in the NSG mouse xenograft model using NPs. Finally, mRNA- and nuclease-loaded NPs were successfully lyophilized for storage, maintaining their transfection potential after rehydration. In conclusion, we show that polymer-based NP delivery of mRNA and nucleases has the potential to overcome current limitations of HSC gene editing. The predictable transfection efficiency, low toxicity, and ability to lyophilize NPs will greatly enhance the portability and provide a highly promising platform for HSC gene therapy.

Keywords: gene editing nucleases; gene therapy; hematopoietic stem cells; lyophilization; polymeric nanoparticles; quality control standards.

Graphical abstract

Figure 1

Characterization and testing of GFP mRNA-loaded NPs on human CD34⁺ cells (A) Cell viability and (B) GFP expression after NP-mediated GFP mRNA delivery. Molar ratios of diacrylate to amine to synthesize polymer batches are indicated at the bottom. Untreated cells were used as a control. (C) GFP expression in NP-treated human CD34⁺ cells (scale bar, 20 μm). (D) Correlation between the transfection efficiency and average size of NP-mRNA. (E) Polydispersity index (PDI) of NP-mRNA. (F) Representative TEM image of NP-mRNA using polymer batch B7 (scale bar, 2 μm; insert, 50 nm). (G) Correlation between the transfection efficiency and the zeta potential of NP-mRNA. Size, PDI, and zeta potential were measured with a Zetasizer. Statistics: (A and B) mean ± SD.

Figure 2

Characterization and testing of RNP-loaded NPs on human CD34⁺ cells (A) Cell viability and knockout of CD33 exon 2 on the cell surface (CD33^ΔE2) determined 72 h after NP-mediated editing of human CD34⁺ cells. The editing efficiency was calculated relative to the background noise in untreated cells. (B) Correlation between the CD33 editing efficiency and size of NP-RNPs. (C) Polydispersity index (PDI) of NP-RNPs. (D) Correlation between the CD33 editing efficiency and zeta potential of NP-RNPs. Statistics: (C) mean ± SD.

Figure 3

Long-term engraftment of NP-RNP-edited human CD34⁺ cells (A–C) Schematic of the experimental design. Longitudinal flow cytometric assessment of (B) human chimerism and (C) CD14⁺ monocytes in the PB. (D) PCR validation of CD33 editing in human cells in the PB at weeks (W) 8 and 10 post-transplant. (E) Quantification of PCR bands in (D). (F) Longitudinal tracking of human CD14⁺ monocytes in the PB lacking CD33 exon 2 on the cell surface (CD33^ΔE2) via flow cytometry. (G) Frequency of human chimerism in the bone marrow (BM), spleen, and thymus. (H) Frequency of engrafted human CD34⁺ cells (left y axis) and the HSC-enriched CD34⁺CD90⁺ subset (right y axis) in the BM. (I) Genomic analysis of the CD33 genotype (+/+, wild type; +/−, heterozygous knockout; −/−, homozygous knockout) of individual CD34⁺- and CD34⁺CD90⁺-derived colonies (representative gel pictures shown in Figure S3I). Statistics: (G and H) mean ± SEM, Wilcoxon signed-rank test.

Figure 4

Long-term peripheral blood chimerism in mice transplanted with NP- versus EP-edited human CD34⁺ cells (A) Cell viability and (B) reduction of CD33 cell-surface expression determined 72 h after NP- and EP-mediated editing of human CD34⁺ cells. Untreated cells were used as a control. (C and D) Longitudinal flow cytometric assessment of (C) human chimerism and (D) CD14⁺ monocytes in the PB in mice transplanted with untreated, EP-RNP-, and NP-RNP-edited human CD34⁺ cells. (E) Human multilineage engraftment in the PB at week 20 post-transplant. (F) PCR validation of CD33 editing in human cells in the PB. (G) Longitudinal monitoring of human CD14⁺ monocytes in the PB lacking CD33 exon 2 on the cell surface (CD33^ΔE2) via flow cytometry. (H) Quantification of PCR bands in (F). Statistics: (A and B) mean ± SD, one-way ANOVA.

Figure 5

Tissue engraftment and reconstitution of the BM stem cell compartment in mice transplanted with NP- versus EP-edited human CD34⁺ cells (A) Frequency of human chimerism in the BM, spleen, and thymus. (B) Frequency of engrafted human CD34⁺ cells (left y axis) and the HSC-enriched CD34⁺CD90⁺ subset (right y axis) engrafted in the BM. (C and D) Total CFC potential (left graphs) and quantification of erythroid, myeloid, and erythromyeloid colonies (right graphs) of human (C) CD34⁺ and (D) CD34⁺CD90⁺ cells. CFU, colony-forming unit; CFU-M, CFU monocyte/macrophage; CFU-G, CFU granulocyte; CFU-GM, CFU granulocyte/monocyte/macrophage; BFU-E, burst-forming unit erythrocyte; CFU-Mix, CFU containing a mix of erythroid and myeloid cells. (E) Quantification of the PCR-based assessment of CD33 exon 2 knockout (CD33^ΔE2) in PB and BM at week 20 shown in Figure S4D. (F) Genomic analysis of the CD33 genotype (+/+, wild type; +/−, heterozygous knockout; −/−, homozygous knockout) of individual CD34⁺- and CD34⁺CD90⁺-derived colonies (representative gel pictures shown in Figures S4E and S4F). Statistics: (A and B) mean ± SEM, one-way ANOVA; (C and D) mean ± SD, one-way ANOVA.

Figure 6

Physicochemical properties and functional testing of lyophilized GFP mRNA- and CD33 RNP-loaded NPs (A) Size and zeta potential of lyophilized NP-mRNAs. (B) Cell viability and transfection efficiency of human CD34⁺ cells treated with either fresh or lyophilized and rehydrated NP-mRNAs (n = 2). (C) Overlay of the previously determined correlation of NP-mRNA charge and transfection efficiency shown in Figure 1F (black dots and line) with the charge of freshly prepared (F) and lyophilized (L) NP-mRNAs. (D) Size and zeta potential of lyophilized NP-RNPs. (E) Cell viability and CD33 exon 2 knockout (CD33^ΔE2) efficiency of human CD34⁺ cells treated with either fresh or lyophilized and rehydrated NP-RNPs (n = 2). (F) Overlay of the previously determined correlation of NP-RNP charge and transfection efficiency shown in Figure 2D (black dots and line) with the charge of freshly prepared (F) and lyophilized (L) NP-RNPs. Statistics: (A, B, D, and E) mean ± SD; (A and D) Wilcoxon signed-rank test.