Triplex-forming PNA design for gene editing

To quantitatively assay for gene editing, we used a mouse model with a β-globin/GFP fusion transgene consisting of human β-globin intron 2 carrying a thalassaemia-associated IVS2-654 (C→T) mutation embedded within the GFP coding sequence, resulting in incorrect splicing of β-globin/GFP mRNA and lack of GFP expression19. PNA-mediated triplex-formation induces recombination of the genomic site with a 60-nucleotide sense donor DNA homologous to the β-globin intron 2 sequence except for providing a wild-type nucleotide at the IVS2-654 position. Correction of the splice-site mutation yields expression of functional GFP (Fig. 1a)12,15, providing a phenotypic read-out of editing quantifiable by flow cytometry.

Figure 1: Gene editing using γPNAs in mouse bone marrow. (a) Strategy for targeted correction of a β-globin gene IVS2-654 (C->T) mutation in β-globin/GFP transgenic mice using triplex-forming tail-clamp PNAs and donor DNAs. (b) tcPNA and γtcPNAs designed to bind to homopurine regions within intron 2 of the human β-globin gene near IVS2-654 (C->T). (c) DNA, unmodified PNA and MPγPNA. (d) tcPNAs and γtcPNAs to bind to the positions indicated in B. γtcPNA4-Scr is a scrambled version of γtcPNA4. Bold/underline indicates γPNA residues. K indicates lysine; J, pseudoisocytosine (for c) for pH-independent triplex formation. O, 8-amino-2,6,10-trioxaoctanoic acid linkers connecting the Hoogsteen and Watson–Crick domains of the tcPNAs. (e) Scanning electron microscope images of nanoparticles. Scale bar, 2.0 μm. (f) Gene correction of the IVS2-654 (C->T) mutation within the β−globin/GFP fusion gene in mouse BM cells treated ex vivo with NPs containing the indicated tcPNAs and donor DNA. %GFP+ was determined by flow cytometry and indicates successful gene editing. Data are mean±s.e., n=3; statistical analysis by Student’s t-test, **P<0.005. (g) %GFP+ cells in mouse BM after ex vivo treatment with NPs containing tcPNA1, γtcPNA4 or γtcPNA4-Scr, plus donor DNAs. Data are mean±s.e.m., n=3; analysis by Student’s t-test, *P<0.05. (h,i) Quantification of γH2AX foci by immune fluorescence microscopy, indicative of DNA DSBs in primary fibroblasts (from the β-globin/GFP transgenic mice) either untreated or treated with 5 Gy of IR, blank NPs, NPs containing γtcPNA4 and donor DNA, lipofectamine alone, lipofectamine transfection of a Cas9 expression vector, lipofectamine transfection of a Cas9 vector and a separate guide RNA expression vector (targeting the same site in β-globin sequences as γtcPNA4; Cas9+gRNA), or transfection of a vector containing both Cas9 and guide RNA (Cas9 and gRNA). Quantification as per cent of cells with 15 or more foci (h) or average number of foci per cell (i). 100 cells were counted per condition, data are mean±s.e.m, n=3; analysis by Student’s t-test, *P<0.05. Full size image

We designed a series of tcPNAs to bind to polypurine stretches in the β-globin intron near the IVS2-654 mutation (Fig. 1b). One of the tcPNAs and a scrambled sequence control were synthesized to contain partial substitution with a mini-polyethylene-glycol group at the γ position (MPγPNA; Fig. 1c,d) within their Watson–Crick binding domains. We made the substitutions in the Watson–Crick domains because in prior work γPNAs have been shown to enhance strand invasion and DNA binding in the Watson–Crick binding mode due to helical pre-organization enforced by the modification16. We hypothesized that this would enhance the binding of the tcPNAs because strand invasion and Watson–Crick PNA/DNA duplex formation is an important component in the formation of PNA/DNA/PNA triplexes. Partial substitution was performed because it is sufficient to improve binding affinity and to confer helical pre-organization20. γtcPNA4 matches the sequence of tcPNA1 except that it contains γ units at alternating positions in the Watson–Crick domain (Fig. 1d). Scrambled γtcPNA (γtcPNA4-Scr) has the same base composition as γtcPNA4 but a scrambled sequence. All tcPNA oligomers were synthesized with 3 lysines at both termini to improve solubility and increase binding affinity (Fig. 1d). Purification and characterization of the synthesized PNAs were performed by high-performance liquid chromatography analyses and matrix-assisted laser desorption/ionization time-of-flight, respectively (Supplementary Fig. 1 and Supplementary Table 1). Gel shift assays to assess the binding of the tcPNAs to DNAs containing the respective target sequences showed that all bound specifically to their target sites (Supplementary Fig. 2A). No binding was seen in the case of the scrambled sequence γtcPNA4-Scr.

In prior work, we showed that poly(lactic-co-glycolic acid) (PLGA) NPs can effectively deliver PNA/donor DNA combinations into primary human and mouse haematopoietic cells with essentially no toxicity12,14,21. Here, tcPNAs and donor DNAs, at a molar ratio of 2:1, were incorporated into poly(lactic-co-glycolic acid) (PLGA) NPs. The NP formulations were evaluated by scanning electron microscopy and dynamic light scattering. All the NPs exhibited sizes within the expected range (Fig. 1e and Supplementary Fig. 2B,C) and showed uniform charge distribution (Supplementary Fig. 2D). Nucleic acid release profiles showed that γ modifications did not impair release from NPs (Supplementary Fig. 2E).

Ex vivo gene editing in bone marrow cells

Bone marrow (BM) cells harvested from β-globin/GFP mice were treated ex vivo with PLGA NPs containing tcPNA1/donor DNA, tcPNA2/donor DNA and tcPNA3/donor DNA. After 48 h, the percentage of GFP+ (corrected) cells was quantified via flow cytometry (Fig. 1f). The higher gene editing activity of tcPNA1 is likely due to its longer Hoogsteen binding domain, an effect that we have previously observed13. NPs containing the γ-substituted tcPNA (γtcPNA4) and donor DNA yielded significantly higher gene modification (1.62%; Fig. 1g and Supplementary Fig. 3A), showing that the MPγ substitutions confers increased biological activity that correlates with their improved binding properties. NPs with the γtcPNA4–Scr produced no modification (Fig. 1g).

BM cells treated with either blank NPs or NPs containing γtcPNA4/donor DNA were plated in methylcellulose medium supplemented with cytokines for growth of granulocyte/macrophage colonies (colony-forming unit (CFU)-G, CFU-M and CFU-GM) or combined colonies (CFU-GEMM, granulocyte, erythroid, monocyte/macrophage and megakaryocyte). The two sets of treated cells formed myeloid and erythroid colonies at similar frequencies (Supplementary Fig. 3B,C), suggesting that treatment with γtcPNA4 and donor DNA does not impair the ability of the progenitor cells to proliferate and differentiate. Sequencing analysis of genomic DNA from selected GFP-positive methylcellulose colonies confirmed the presence of the targeted gene modification in the β-globin/GFP transgene at the IVS2-654 base pair (Supplementary Fig. 3D). Also, there was no induction of the inflammatory cytokines in the treated BM cells (Supplementary Fig. 3E), consistent with prior work with NPs containing standard PNAs12,14,21.

In assays for genotoxicity, there was no detectable increase in DNA double-strand breaks (DSBs) in the BM cells treated with γtcPNA4/donor DNA-containing NPs compared with blank NPs based on a single-cell gel electrophoresis assay (Comet assay; Supplementary Fig. 4A). We also assayed for DSBs by measuring the production of γH2AX foci. γH2AX foci are detected in nuclei by immune fluorescence and document a chromatin modification that occurs upon DSB formation via DNA damage response signalling22. Treatment of the BM cells with NPs containing γtcPNA4/donor DNA did not produce any γH2AX foci above the low background that is seen in untreated cells (assayed by flow cytometry as shown in Supplementary Fig. 4B). Treatment of the cells with 5 Gy of ionizing radiation (IR) was included as a positive control for induction of DSBs. We also used primary fibroblasts from the mice for this assay, since adherent cells facilitate more robust quantification of foci by immune fluorescence microscopy. Treatment of the fibroblasts with NPs containing γtcPNA4/donor DNA again did not produce γH2AX foci above the low background in untreated or blank NP treated cells (quantified in Fig. 1h as per cent of cells with more than 15 foci and in Fig. 1i as average number of foci per nucleus, with corresponding immune fluorescence images of foci shown in Supplementary Fig. 5). For comparison, transfection of a vector expressing the Cas9 nuclease yielded an increase in γH2AX foci (Fig. 1h,i). Co-expression of a guide RNA designed to bind to the same sequence in the β-globin gene as γtcPNA4 (either via the same plasmid or via a plasmid separate from the Cas9 vector) reduced the number of induced DSBs, but to a level that was still above background (Fig. 1h,i and Supplementary Fig. 5).

Elevated gene editing by γtcPNAs in CD117+ cells

Previous work suggested that there might be increased PNA-mediated editing in colony-forming progenitors12. To test this, we treated whole BM cells with either blank NPs, NPs containing tcPNA1/donor DNA, or NPs containing γtcPNA4/donor DNA. Two days later, flow cytometry was performed to assess the frequency of GFP+ cells within selected sub-populations. CD117+ cells showed elevated gene editing compared with the total CD45+ cell population (Fig. 2a), with 8.6% in CD117+ cells after a single treatment with γtcPNA4/donor DNA NPs. The less potent tcPNA1/donor DNA NPs still yielded an elevated correction frequency of 2.1% in CD117+ cells compared with the total BM (although fourfold lower than γtcPNA4). Next, we sorted for CD117+ cells before treatment with the NPs (Fig. 2b). An elevated percentage of modification (7.2%) was again seen after a single treatment (Fig. 2b).

Figure 2: The SCF/c-Kit+ pathway promotes gene editing and DNA repair. (a) %GFP expression in treated mouse BM cells expressing the indicated cell surface markers. Total BM was treated with NPs containing either tcPNA1/donor DNA or γtcPNA4/donor DNA, cells were stained using antibodies specific for the indicated markers and assayed by flow cytometry for marker and GFP expression. Data are mean±s.e.m, n=3; statistical analysis by Student’s t-test, *P<0.05. (b) %GFP expression in pre-sorted CD117 (c-Kit+) cells treated with either NPs carrying γtcPNAs and donor DNAs or blank NPs. Data are mean±s.e.m., n=3; statistical analysis by Student’s t-test, **P<0.005. (c) %GFP expression in pre-sorted CD117+ cells treated with NPs containing γtcPNA4/donor DNA with or without prior treatment with the c-Kit ligand, SCF. Data are mean±s.e.m., n=3; statistical analysis by Student’s t-test, *P<0.05. (d) %GFP expression in pre-sorted CD117+ cells treated with NPs containing γtcPNA4/donor DNA in the presence or absence of c-Kit pathway kinase inhibitors: dasatinib (inhibits c-Kit), MEK162 (inhibits MEK, MEK) and BKM120 (inhibits phosphatidylinositol-3-kinase). Data are mean±s.e.m, n=3; statistical analysis by Student’s t-test, *P<0.05. (e) Heat map showing upregulated genes involved in DNA repair in CD117+ cells with or without SCF treatment; rows are clustered by Euclidean distance measure. (f) Reporter gene assay for HDR activity in CD117 cells in the presence or absence of c-Kit pathway kinase inhibitors: dasatinib, MEK162 and BKM120 (as above). Inset shows diagram of the luciferase reporter assay for repair of a nuclease-indcued DSB by HDR. Luciferase expression occurs only after homologous recombination and is scored as % reactivation of the DSB-damaged plasmid, normalized to a transfection control. Data are mean±s.e.m., n=3; statistical analysis by Student’s t-test, ***P<0.005. (g) HDR assay in CD117+ cells with or without the addition of SCF. Data are shown as mean±s.e.m., n=3; statistical analysis by Student’s t-test, ***P<0.005 and ****P<0.0005. Full size image

The c-Kit pathway mediates increased gene editing

CD117 (also known as mast/stem cell growth factor receptor or proto-oncogene c-Kit protein) is a receptor tyrosine kinase expressed on the surface of haematopoietic stem and progenitor cells and other cell types. SCF, the ligand for c-Kit, causes dimerization of the receptor, activating its kinase activity to trigger signalling pathways that impact survival, proliferation and differentiation.

We asked whether c-Kit-dependent signalling is required for elevated gene correction or whether CD117 simply serves as a marker for the increased susceptibility. First, we tested for gene editing in pre-sorted CD117+ cells treated with or without the c-Kit ligand, SCF, and we observed a significant increase in γtcPNA4/donor DNA-mediated gene editing (up to almost 15%) in the SCF-treated cells (Fig. 2c and Supplementary Fig. 6). We next assayed for γtcPNA4/donor DNA NP-mediated gene editing in pre-sorted CD117+ cells in the presence or absence of kinase inhibitors (Fig. 2d). Dasatinib, which inhibits the c-Kit kinase in addition to the BCR/Abl and Src kinases, reduced the gene editing from 7 to 2.0%. Inhibitors of factors downstream of c-Kit, including mitogen/extracellular signal-regulated kinase (MEK; Binimetinib; MEK162) and phosphatidylinositol-3-kinase (BKM120), also decreased gene editing in CD117+ cells to 2.6 and 4.1%, respectively (Fig. 2d).

We also tested tcPNA1 (no gamma substitutions) for gene editing in pre-sorted CD117 cells treated or not with SCF. We found that SCF boosted tcPNA1/donor DNA gene editing up to 5% versus about 1.5% without SCF (Supplementary Fig. 7). While substantial, this was threefold lower than the almost 15% frequency seen with the γtcPNA4/donor DNA NPs in conjunction with SCF treatment of the pre-sorted CD117 cells (Fig. 2c). Overall, these results indicate that the SCF/c-Kit signalling can enhance gene editing and identify SCF as a potential agent to stimulate PNA-mediated gene modification. In addition, the results further show that γtcPNAs provide increased gene editing potential as compared with regular tcPNAs.

Increased DNA repair gene expression upon activation of c-Kit+

To explain the increased gene editing in CD117 cells, we tested whether CD117 might be associated with differential uptake of the NPs. However, there were no differences in uptake across several BM cell sub-populations (Supplementary Fig. 8). Next, we examined gene expression patterns in the c-Kit+ cells to test the hypothesis that there might be increased DNA repair gene expression. RNA from sorted CD117+ and CD117− cells from the β-globin/GFP mice was analysed using Illumina arrays (Supplementary Fig. 9A–C). We found that numerous DNA repair genes, including BRCA1, BRCA2, Rad51, ERCC2, XRCC2 and XRCC3, showed higher levels of expression in CD117+ cells (Supplementary Figs 10 and 11a)., The elevated expression two genes likely to play a role in PNA-induced recombination, BRCA2 and Rad51, was confirmed in CD117+ cells by quantitative PCR with reverse transcription (Supplementary Fig. 11B,C) and by western blot (Supplementary Fig. 11D). We went on to test whether SCF treatment could further increase DNA repair gene expression. Gene expression profiling showed additional up-regulation of numerous DNA repair genes by SCF treatment (Fig. 2e and Supplementary Fig. 12A,B).

Because SCF, as a stimulatory cyctokine, would be expected to impact the cell cycle in c-Kit+ cells and because HDR gene expression is known to be elevated in S-phase, we also measured the effect of SCF on cell cycle phase distribution in the pre-sorted CD117 cells. We found that there was a 9% increase in the per cent of cells in S-phase at 48 h after SCF treatment as compared with no SCF treatment (Supplementary Fig. 13). Hence, some component of the SCF effect on DNA repair genes could be an indirect effect of cell cycle changes.

The c-Kit pathway induces functionally elevated DNA repair

To test whether the above increases in DNA repair gene expression could be correlated with functional differences in DNA repair, we used a luciferase-based assay to quantify repair of DNA DSBs by HDR. In this assay, repair of a DSB in a reporter plasmid via intramolecular homologous recombination creates (‘reactivates’) a functional luciferase gene (Fig. 2f), and so the assay provides a measure of HDR capacity (validation of the assay is shown in Supplementary Fig. 14). The results show increased HDR in CD117+ compared with CD117− cells (Fig. 2f). HDR in CD117+ cells was diminished by the kinase inhibitors MEK162, BKM120 and dasatnib (Fig. 2g); conversely, it was boosted by SCF treatment (Fig. 2g). These results indicate that c-Kit signalling increases HDR.

In vivo gene editing by PNA/DNA NPs given intravenously

We next tested the potential for in vivo gene editing in the β-globin/GFP transgenic mice by simple intravenous injection of NPs containing tcPNA1/donor DNA or γtcPNA4/donor DNA, and we further asked whether the editing could be enhanced by SCF treatment. Mice were treated with a single dose of 4 mg NPs, and 2 days later we analysed gene editing in BM and spleen. Some mice also received murine SCF (220 μg kg−1) given by intraperitoneal injection 3 h before the NP injection (Fig. 3a,b, with experimental scheme shown in Supplementary Fig. 15A). In vivo gene editing was scored by GFP expression in marker-sorted cell populations from BM and spleen (Fig. 3a,b). We observed an average of ∼0.2% gene editing in the CD117 BM cells from tcPNA1/donor DNA and SCF-treated mice (Fig. 3a). Two to threefold higher levels of gene editing were seen in CD117+ cells from BM of the γtcPNA4/donor DNA and SCF-treated mice, with frequencies in the range of 1% in several mice, and average frequencies in the 0.5% range. Similar frequencies were seen in spleen (Fig. 3b).

Figure 3: SCF treatment of mice enhances in vivo gene editing by γtcPNAs and donor DNAs. (a,b) β-globin/GFP transgenic mice (n=6 mice per group) were injected or not (as indicated) with 220 μg kg−1 of SCF i.p. followed by a single treatment of 4 mg of NPs injected intravenously. Each group received either blank NPs, NPs containing tcPNA1 and donor DNA, or NPs containing γtcPNA4 and donor DNA, with or without SCF. Two days later, BM and spleen cells were harvested for analysis by flow cytometry and deep sequencing. Frequencies of gene editing in haematopoietic cell sub-populations identified by the indicated cell surface markers from BM (a) and spleen (b) of mice treated with the indicated NPs and with or without pre-treatment with SCF. Each data point represents analysis of cells from a single mouse. Horizontal bars indicate mean, statistical analysis by Student’s unpaired t-test, *P<0.05. (c) Deep-sequencing analysis to quantify the frequency of targeted gene editing in vivo in CD117+ cells from BM and spleen of β-globin/GFP mice treated as above. Error bars indicate standard error of proportions. (d) Analysis of cytokine levels, as indicated, in blood of either untreated, blank NP treated, or γtcPNA4 and donor DNA NP treated mice at 48 h post treatment. Data are shown as mean±s.e.m., n=3. Full size image

We confirmed these results by performing deep sequencing analysis on DNA from CD117+ cells isolated from BM and spleen of treated mice (Fig. 3c and Supplementary Fig. 15A), revealing gene editing frequencies in the range of 0.2% in the BM of mice treated with γtcPNA4/donor DNA NPs without SCF and 0.6% in mice receiving SCF along with the γtcPNA4/donor DNA NPs (in a single treatment in each case), consistent with the frequencies of gene correction quantified by GFP expression. Deep sequencing was also used to assess off-target effects in the BM cells of the mice that were treated with SCF and γtcPNA4 and donor DNA NPs (Table 1). By BLAST analysis, we identified seven off-target sites with partial homology to the target site of γtcPNA4 in β-globin intron 2. Extremely low frequencies of off-target effects were found in the γtcPNA4/donor DNA treated mice, with six sites showing no detectable sequence changes out of millions of reads and two sites showing modification frequencies of only 0.0074 and 0.00018% compared with 0.56% at the targeted β-globin site (Table 1). The overall off-target modification frequency at all seven sites combined was 0.00034%, 1,647-fold lower than the frequency of the targeted gene editing.

Table 1 Off-target effects in bone marrow cells following intravenous treatment of β-globin/GFP mice with γtcPNA4/donor DNA NPs. Full size table

We also performed cytokine array analyses on plasma derived from mice 48 h after treatment with γtcPNA4/donor DNA NPs . There were no detectable increases in levels of any of the cytokines measured compared with untreated mice (Fig. 3d), with lipopolysaccharide treatment as a positive control showing significantly higher levels of multiple cytokines that were off the scale of the graph (Supplementary Fig. 16). In a separate experiment, mice were treated with γtcPNA4/donor DNA NPs on day 1 and again 3 months later, followed by cytokine analysis of peripheral blood, again showing minimal immune or inflammatory response (Supplementary Fig. 17A,B).

In vivo correction of a β-thalassaemia mutation in mice

We next tested for correction of a human β-thalassaemia mutation in a mouse disease model, using a transgenic mouse line in which the two (cis) murine adult beta globin genes were replaced with a single copy of the human β-globin gene with the same thalassaemia-associated IVS2-654 mutation as above18. Homozygous mice do not survive, and heterozygotes have a moderate form of β-thalassaemia, with haemolytic anaemia, microcytosis and other erythrocyte morphologies reflecting reduced amounts of mouse β-globin and no human β-globin17,18, consistent with β-thalassaemia. Treatment groups included (1) blank NPs; (2) SCF alone (no NPs); (3) SCF plus γtcPNA4/donor DNA NPs; and (4) SCF plus γtcPNA4-Scr/donor DNA (experimental scheme is shown in Supplementary Fig. 15B). We conducted two otherwise identical replicate experiments except that, in one, the complete blood count (CBC) analyses were continued for 75 days after the last treatment, and in the other for 140 days. In both cases, each treatment group consisted of six mice, and each mouse received four treatments at 2 day intervals at the beginning of the experiment as indicated in Supplementary Fig. 15B. Some animals were maintained long-term for serial CBC analyses; others were killed at intermediate time points for analysis of spleen size and architecture and for deep sequencing of BM-derived cells.

Blood smears at day 0 (before treatment) and at day 36 after the last treatment (Fig. 4a) showed marked improvement in RBC morphology on day 36 in the γtcPNA4/donor DNA treated mice but not in the mice treated with either blank NPs, SCF alone or SCF plus γtcPNA4-Scr/donor DNA. CBC analyses performed on blood samples taken at 30, 45, 60, 75, 90 and 140 days post-treatment from mice in each group showed persistent correction of the anaemia based on blood haemoglobin levels in the mice treated with SCF plus the γtcPNA4/donor DNA NPs (Fig. 4b), with elevation of the blood haemoglobin levels into the normal range. The anaemia was not improved in any of the controls. We also observed reduced reticulocyte counts in mice treated with SCF plus the γtcPNA4/donor DNA NPs but not in the mice treated with blank NPs (Fig. 4c). In addition, the γtcPNA4/donor DNA treated mice also showed reduced splenomegaly at 36 days post-treatment (Fig. 4d).

Figure 4: Correction of anaemia in thalassemic mice by NPs containing γtcPNA and donor DNA. (a) Blood smears from wild-type and thalassemic mice obtained pre-treatment or 36 days after in vivo treatment with blank NPs, SCF alone, SCF plus scrambled γtcPNA4-Scr/donor DNA NPs or SCF plus γtcPNA4/donor DNA NPs. NPs were given i.v.; SCF i.p. The untreated group (and control animals) exhibit extreme poikilocytosis as well as numerous target cells, cabot rings, anisochromasia and ovalocytosis, all changes characteristic of β-thalassemia. Treatment with γtcPNA4/donor DNA and SCF ameliorates the poikilocytosis and yields a reduction in anisocytosis, ovalocytosis and target cells suggestive of reduced alpha-globin precipitation in the RBCs. Scale bar, 1.0 μm. (b) Blood haemoglobin levels of thalassemic mice treated with blank NPs, SCF plus scrambled γtcPNA4-Scr/donor DNA NPs, or with SCF plus γtcPNA4/donor DNA NPs performed at the indicated number of days after treatment, up to 140 days. Data are presented as box and whisker plots showing the median and quartile range within each group over time (n=6 per group). Only the SCF plus γtcPNA4/donor DNA-treated mice achieved and maintained haemoglobin levels within the normal range during the duration of the experiment, reflecting the increased haemoglobin stability conferred by the gene editing. Horizontal bars within the boxes indicate mean; statistical analysis by Student’s unpaired t-test, *P<0.05. (c) Reticulocyte counts (% of total RBCs) calculated in blood smears from thalassemic mice treated with either blank NPs or with NPs containing and γtcPNA4/donor DNA plus SCF on days 0 and 36 post treatment. Data are mean±s.e.m., n=3; statistical analysis by Student’s t-test, *P<0.05. (d) Images of spleens from wild-type mice or thalassemic mice treated with blank NPs, SCF alone, SCF plus scrambled γtcPNA4-Scr/donor DNA NPs or SCF plus γtcPNA4/donor DNA NPs at 36 days after the last treatment. Average spleen weights (with standard errors, n=3; except for wild-type, where n=1) are listed below the images. Splenomegaly is corrected only by SCF plus γtcPNA4/donor DNA treatment. Scale bar, 1.0 cm. (e) Histopathologic analysis of spleen sections from wild-type mice and from thalassemic mice obtained 36 days after treatment with blank NPs or SCF plus γtcPNA4/donor DNA NPs. Haematoxylin and eosin stain (H&E), × 40 magnification. Scale bar, 10.0 μm. Additional images of spleens from other treatment groups and with additional stains are presented in Supplementary Figs 17 and 18. Full size image

Consistent with reduced splenomegaly, histologic examination of spleens from mice on days 36 and 75 showed substantially improved splenic architecture specifically in the γtcPNA4/donor DNA treated mice (Fig. 4e and Supplementary Figs 18 and 19). The regular splenic pattern of white pulp (lymphoid follicles) surrounded by rims of red pulp is disrupted in the β-thalassemic animals due to extramedullary haematopoiesis, causing an expansion in red pulp (causing the splenomegaly) and disruption of the white pulp. The CD61 and Ecad immunohistochemical stains (Supplementary Fig. 18) highlight the increased cellularity characteristic of extramedullary haematopoiesis and demonstrate that the expanded red pulp includes elevated megakaryocytes and erythroid precursors, respectively. This increased cellularity is substantially ameliorated in the γtcPNA4/donor DNA treated mice (Supplementary Fig. 18).

Deep sequencing analyses were performed on total BM cells of mice on day 36 post-treatment. Correction of the targeted mutation was seen at a frequency of almost 4% in the γtcPNA4/donor DNA treated group (Fig. 5a; combined analysis of 3 mice), whereas no correction was seen in the mice treated with blank NPs (Fig. 5a). Deep sequencing was also used to assess off-target effects in the BM cells at seven sites with partial homology to the binding site of γtcPNA4 in the β-globin gene. We found extremely low frequencies of off-target effects in the γtcPNA4/donor DNA-treated thalassemic mice (Table 2). The overall off-target modification frequency was 0.0032%, 1,218-fold lower than the frequency of β-globin gene editing.

Figure 5: Gene editing in mouse BM stem cell populations in vivo and human CD34+ cells ex vivo. (a) Thalassemic mice were treated with either blank NPs or NPs containing γtcPNA4/donor DNA plus SCF (four doses at 2-day intervals as in Fig. 4). Deep sequencing analysis was performed to measure gene editing in the β-globin gene in either total BM (total BM) cells or in BM stem/progenitor cell sub-populations selected based on the indicated markers. BM cells were harvested either on day 36 post-treatment (total BM samples) or on day 65 post-treatment (sorted cell sub-populations). Data represent the combined analysis of BM from n=3 mice in each group. Data are mean±s.e.m., n=3; statistical analysis by Student’s t-test, *P<0.05. (b) Schematic showing experimental design in which human CD34+ cells were treated ex vivo with either blank NPs or with γtcPNA4/donor DNA NPs plus SCF. Treated cells were either harvested 2 days later for deep sequencing analysis of gene editing in the β-globin gene or were transplanted into NOD-scid IL2rγnullmice. Eight weeks after transplantation, BM cells were harvested from the mice and human CD34+ cells were isolated, followed by deep sequencing of the the β-globin gene alleles. (c) Deep sequencing results to quantify β-globin gene editing in either pre-transplanted human CD34+ cells or in human CD34+ cells that were harvested from NOD-scid IL2rγnull mice 8 weeks after transplant, as described in b. Data are mean±s.e.m., n=3 statistical analysis by Student’s t-test, *P<0.05. Full size image

Table 2 Off-target effects in bone marrow cells following intravenous treatment of β-thalassemic mice with SCF and γtcPNA4/donor DNA NPs. Full size table

In addition, we sorted the cells from the BM of the SCF and γtcPNA4/donor DNA NP treated mice (the BM was collected 65 days after the last treatment) for markers consistent with several stem/progenitor cell populations23, and we again performed deep sequencing of the β-globin gene. This revealed evidence for gene editing at a frequency of 6.9% in Lin-Sca1+ cKit+ CD150+ CD135-cells (Fig. 5a and Supplementary Fig. 20), a population that is highly enriched for long-term HSCs. We also observed gene editing in multiple other progenitor populations (Fig. 5a).

We also noted that the β-thalassemic animals had elevated levels of CD117+ cells in their BM compared with the phenotypically normal β-globin/GFP transgenic mice (Supplementary Fig. 21), indicative of the stress erythropoeisis in these animals. We also found that the proportion of CD117+ cells was further increased by SCF treatment. These elevated levels of CD117+ cells, and the stress erythropoeisis that underlies them, could explain, in part, the increased susceptibility to gene editing in the thalassemic mice.

Gene editing by γtcPNAs in CD34+ human HSCs

We next tested the gene editing potential of γtcPNA4/donor DNA NPs in human CD34+ cells. Because we did not have cells from a thalassemic patient, we obtained human CD34+ cells from healthy donors from a cell bank. Consequently, we used a modified donor DNA designed to introduce a mutation at position IVS2-654 rather than correct it. One day after the CD34+ cells were thawed into stem cell medium, they were treated either with blank NPs or with NPs containing γtcPNA4/donor DNA plus SCF. Two days later, we performed deep sequencing, revealing β-globin gene editing at position IVS2-654 at a frequency of 5.0% (Fig. 5b,c and Table 3). Six off-target sites with partial homology to the binding site of γtcPNA4 in the β-globin gene were also analysed, and extremely low off-target frequencies were found in two of the sites sites (0.000017 and 0.000055%), with four sites showing no detectable sequence changes out of millions of reads (Table 3). Combined, we saw off-target mutations at an overall frequency of only 0.000012%, more than 400,000-fold lower than the 5% frequency of editing in the targeted β-globin gene. In addition, cytokine array analysis of supernatant taken from the treated CD34+ cells showed a minimal cytokine response (Supplementary Fig. 22).

Table 3 Deep sequencing analysis of targeted gene editing versus off-target effects in human CD34+ haematopoietic cells following ex vivo treatment with SCF and γtcPNA4/donor DNA NPs. Full size table

We also performed a transplantation experiment in which human CD34+ cells that were treated ex vivo with γtcPNA4/donor DNA NPs and SCF were then transplanted into NOD-scid IL2rγnullmice. Eight weeks later, human CD34+ cells were isolated from the BM of the mice and deep sequencing was performed to measure the presence of gene editing in the β-globin gene (Fig. 5b). We found that 3.4% of β-globin gene alleles showed the introduced mutation at position IVS2-654 (Fig. 5c).