Development of a cell-penetrating peptide-based nanocomplex for long-term delivery of intact mitochondrial DNA into epithelial cells

Abstract

Gene therapy approaches for mitochondrial DNA (mtDNA)-associated damage/diseases have thus far been limited, and despite advancements in single gene therapy for mtDNA mutations and progress in mitochondrial transplantation, no method exists for restoring the entire mtDNA molecule in a clinically translatable manner. Here, we present for the first time a strategy to deliver an exogenous, fully intact, and healthy mtDNA template into cells to correct endogenous mtDNA mutations and deletions, with the potential to be developed into an efficient pan-therapy for inherited and/or acquired mtDNA disorders. More specifically, the novel therapeutic nanoparticle complex used in our study was generated by combining a cell-penetrating peptide (CPP) with purified mtDNA, in conjunction with a mitochondrial targeting reagent. The generated nanoparticle complexes were found to be taken up by cells and localized to mitochondria, with exogenous mtDNA retention/maintenance, along with mitochondrial RNA and protein production, observed in mitochondria-depleted ARPE-19 cells at least 4 weeks following a single treatment. These data demonstrate the feasibility of restoring mtDNA in cells via a CPP carrier, with the therapeutic potential to correct mtDNA damage independent of the number of gene mutations found within the mtDNA.

Keywords: MT: Delivery Strategies; age-related diseases; cell-penetrating peptide; mitochondria; mitochondrial DNA; mitochondrial transplantation; mtDNA; mtDNA gene therapy; mtDNA mutations; nucleic acid delivery.

Graphical abstract

Figure 1

Fabrication and characterization of mtDNA nanoparticles Nanoparticle complexes were formed by mixing RD3AD and mtDNA/Rho123 at different N:P molar ratios (0 [i.e., mtDNA/Rho123 only], 0.225, 1.125, 2.25, 4.5, 6.75, 9, and 11.25). (A) Representative gel image of nanoparticle complexes electrophoresed on an agarose gel. mtDNA/Rho123 was observed as having migrated through the gel (indicated by arrow), while peptide-bound mtDNA remained trapped in the well. (B) Bands from gel shift assays were quantified with ImageJ software and compared against mtDNA/Rho123 to determine peptide binding efficiencies. Data are mean ± SEM of three independent samples, where ∗∗∗∗p < 0.0001 and p ≥ 0.05 is not significant (ns) compared to mtDNA/Rho123 (one-way ANOVA with Dunnett’s multiple comparisons test). (C and D) Particle sizes (C, diameter [nm]) and particle charges (D, zeta potential [mV]) of complexes as quantified by nanoparticle tracking analysis at 1.125, 3, and 11.25 N:P molar ratios. Data are mean ± SEM of three independent samples, where ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and p ≥ 0.05 is ns.

Figure 2

Nanoparticle visualization using atomic force microscopy Atomic force microscope deflection error images (10 × 10 mm) of (A) mtDNA/Rho123 complex mixed 1:1 with 100% EtOH, (B) RD3AD/mtDNA/Rho123 nanocomplex (N:P molar ratio of 1.125) mixed 1:1 with 100% EtOH, and (C) RD3AD/mtDNA/Rho123 nanocomplex (N:P molar ratio of 11.25) mixed 1:1 with 100% EtOH.

Figure 3

mtDNA protection by and release from the RD3AD peptide (A) Representative gel image of undegraded mtDNA (indicated by arrow) from mtDNA/Rho123 or RD3AD/mtDNA/Rho123 (N:P molar ratio of 11.25) complexes incubated with or without DNase I. (B) Quantification of mtDNA bands after DNase I treatment of the RD3AD/mtDNA/Rho123 nanocomplex compared against an untreated sample to determine protection efficiency. Data are mean ± SEM of three independent samples, where p ≥ 0.05 is not significant (ns) compared to untreated samples (unpaired t test). (C) Representative gel image of released mtDNA (indicated by arrow) from RD3AD/mtDNA/Rho123 (N:P molar ratio of 11.25) complexes following incubation with increasing amounts of heparin. An untreated mtDNA/Rho123 complex was used to demarcate the free mtDNA within the gel. (D) Quantification of the amount of mtDNA displaced from the RD3AD/mtDNA/Rho123 nanocomplex upon heparin challenge compared against an untreated mtDNA/Rho123 complex to determine percentage of release. Data are mean ± SEM of four independent samples, where ∗∗p < 0.01, ∗∗∗∗p < 0.0001, and p ≥ 0.05 is ns.

Figure 4

Nanoparticle cell uptake and mitochondrial localization (A) Live cell imaging of ARPE-19 cells, pre-stained with MitoTracker Green and Hoechst dyes, following nanocomplex treatment at 0 h (top: RD3AD/mtDNA-Cy5/Rho123, bottom: CPP-imp/mtDNA-Cy5/Rho123). Images were then taken separately at 405 (Hoechst), 488 (MitoTracker Green), and 640 (Cy5) nm over a 4-h time course and processed to show nuclei (blue), mitochondria (magenta), mtDNA-containing nanocomplexes (green), and nanocomplex and mitochondrial colocalization (white). To better visualize the degree of colocalization, mitochondrial networks were outlined, and colocalization of nanoparticles and mitochondria were highlighted in red (see insets). Scale bar: 5 μm. (B) Cross-sectional imaging of fixed ARPE-19 cells (pre-stained with MitoTracker Green and Hoechst dyes) 4 h post-treatment with the RD3AD/mtDNA-Cy5/Rho123 nanocomplex. Center x-y is the focal plane (scale bar: 5 μm). The first three rows are the view of the outlined area in the x-z direction (scale bar: 2 μm), while the three rows to the right are the view of the outlined area in the y-z direction (scale bar: 2 μm). (C) Representative gel images of mtDNA-encoded ND1 gene DNA detection via PCR from cytosolic and mitochondrial fractions of ρ0 cells 24 h post-treatment with RD3AD/mtDNA or RD3AD/mtDNA/Rho123 complexes compared to untreated cells. β-actin (β-act) was used as a normalizing control. (D) Quantification of the levels of ND1 gene DNA present in mitochondrial and cytosolic fractions of ρ0 cells as obtained in (C). All values were normalized to the untreated mitochondrial fraction for direct comparisons. Data are mean ± SEM of four independent samples, and values were compared to the untreated samples (two-way ANOVA), where ∗p < 0.05 and p ≥ 0.05 is ns.

Figure 5

Nanoparticle toxicity TEER values at 0 (pre-treatment), 4, 24, and 48 h following treatment of polarized monolayers of (A) ρ0 and (B) ARPE-19 cell polarized monolayers with mtDNA/Rho123 or RD3AD/mtDNA/Rho123 complexes, or 5 mM H₂O₂. The values for all time points were normalized to the 0-h time point for each treatment. Data are mean ± SEM of four independent samples, and values were compared to the untreated sample in each time point (two-way ANOVA), where ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, and p ≥ 0.05 is not significant. (C and D) MTS assays were performed on the same ρ0 (C) and ARPE-19 (D) cells after the 48-h TEER reading was taken. Absorption readings were measured and compared to untreated cells for each treatment group. Data are mean ± SEM of four independent samples, and values were compared to the untreated samples in each time point (one-way ANOVA), where ∗∗p < 0.01, ∗∗∗∗p < 0.0001, and p ≥ 0.05 is not significant. We note that 5 mM H₂O₂ served as a positive control for cytotoxicity.

Figure 6

mtDNA transfer and function after 4 weeks following single treatment (A) Schematic of overlapping primers for long-range PCR for the detection of mtDNA and verification of primer efficiency in healthy ARPE-19 cells.(B) Representative gel image of intact mtDNA detection using long-range PCR from harvested ρ0 cell polarized monolayers untreated or treated for 4 weeks with mtDNA/Rho123, RD3AD/mtDNA (N:P molar ratio of 11.25), or RD3AD/mtDNA/Rho123 (N:P molar ratio of 11.25) complexes. (C) Representative gel image of intact mtDNA detection using long-range PCR from harvested ρ0 cell polarized monolayers untreated or treated for 4 weeks with 210 μg freshly isolated ARPE-19 mitochondria (containing ∼1.8 μg mtDNA) or the RD3AD/mtDNA/Rho123 nanocomplex (N:P molar ratio of 11.25; containing ∼2 μg mtDNA). (D) PCR bands from (C) were quantified with ImageLab and then normalized to the mitochondrial treated sample for each primer set. Data are mean ± SEM of three biological replicate samples, and values were compared to the transplanted mitochondria treatment group (ANOVA), where ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and p ≥ 0.05 is not significant. (E) TaqMan multiplex quantitative real-time PCR analysis of the mitochondrial RNA for the RNR2 gene (mtRNR2), normalized to the β-actin transcript, 4 weeks following nanocomplex treatment of ρ0 cells. Data were analyzed following the 2^−ΔΔCT method, with three technical replicates for each biological replicate. Data are mean ± SEM of three biological replicate samples, and values were compared to the untreated group (unpaired t test), where ∗p < 0.05 and p ≥ 0.05 is not significant. (F) Western blot analyses of mitochondrial protein MT-CO2 at 4 weeks following nanocomplex treatment of ρ0 cells. Samples were re-probed for GAPDH as a control for equal loading. Bands were quantified using ImageJ software, and data are mean ± SEM of five biological replicate samples, with the values compared to the untreated group (unpaired t test). ∗p < 0.05 and p ≥ 0.05 is not significant.