Correction of pathogenic mitochondrial DNA in patient-derived disease models using mitochondrial base editors

Abstract

Mutations in the mitochondrial genome can cause maternally inherited diseases, cancer, and aging-related conditions. Recent technological progress now enables the creation and correction of mutations in the mitochondrial genome, but it remains relatively unknown how patients with primary mitochondrial disease can benefit from this technology. Here, we demonstrate the potential of the double-stranded DNA deaminase toxin A-derived cytosine base editor (DdCBE) to develop disease models and therapeutic strategies for mitochondrial disease in primary human cells. Introduction of the m.15150G > A mutation in liver organoids resulted in organoid lines with varying degrees of heteroplasmy and correspondingly reduced ATP production, providing a unique model to study functional consequences of different levels of heteroplasmy of this mutation. Correction of the m.4291T > C mutation in patient-derived fibroblasts restored mitochondrial membrane potential. DdCBE generated sustainable edits with high specificity and product purity. To prepare for clinical application, we found that mRNA-mediated mitochondrial base editing resulted in increased efficiency and cellular viability compared to DNA-mediated editing. Moreover, we showed efficient delivery of the mRNA mitochondrial base editors using lipid nanoparticles, which is currently the most advanced non-viral in vivo delivery system for gene products. Our study thus demonstrates the potential of mitochondrial base editing to not only generate unique in vitro models to study these diseases, but also to functionally correct mitochondrial mutations in patient-derived cells for future therapeutic purposes.

Fig 1. Creation of m.15150G > A using DdCBE in human liver organoids.

(A) Design of two sets of Left (red) and Right (green) target sequences flanking the spacer region (blue) for TALE-guided editing in MT-CYB to create m.15150G > A (dark red). (B) Editing efficiencies of all four combinations of Left- and Right-DdCBE plasmids in HEK293T cells as determined by Illumina Next Generation Sequencing (NGS). L1- or L2-only were used as negative control. N = 3, one-way ANOVA with Tukey’s multiple comparisons post hoc test. (C) Ratio of on-target editing vs. bystander editing at any other base within the spacing region in HEK293T cells. N = 3, Welch’s t test. (D) Top: Reduced MT-CYTB protein expression in two technical replicates of m.15150G > A-edited HEK293T cells. Bottom: quantification of MT-CYTB bands relative to HSP90 as analyzed by densitometry. N = 2. (E) Overview of the transfection strategy of DdCBE for organoids. (F) Editing efficiencies of L1R1 constructs modRNA in liver organoids, Sanger sequencing N = 3, Welch’s t test. (G) Clonal lines of edited organoids show high variability in mitochondrial heteroplasmy, indicating different editing efficiencies (Illumina NGS) per cell. Variance-to-mean ratios (VMRs) are displayed above each replicate (N = 12 clones for both replicates). (H) ATP levels decrease in organoids containing 24% m.15150G > A. N = 3, Paired t test. Raw data are provided in S1 Data.

Fig 2. Correction of m.4291T > C in primary patient-derived fibroblasts.

(A) The primary patient-derived fibroblast line is homoplasmic for the m.4291T > C mutation. (B) Design of two sets of Left (red) and Right (green) target sequences flanking the spacer region (blue) for TALE-guided editing in MT-TI to correct m.4291C > T (dark red). Mismatched T (red) in TALE target sequence L2. (C) Overview of the transfection strategy of DdCBE for primary skin fibroblasts. (D) Editing efficiencies of all four combinations Left- and Right-TALE-DdCBE plasmids in primary patient-derived fibroblasts as measured by amplicon sequencing (Illumina NGS). Transfection with Left-only DdCBE plasmid was used as negative control. N = 5 for L1/L2 and L2R2, N = 4 for L1R1 and L2R1, N = 3 for L1R2; one-way ANOVA with Tukey’s multiple comparisons post hoc test. (E) Ratio of on-target editing vs. bystander editing at any other base within the spacing region in fibroblast cells. One-way ANOVA with Tukey’s multiple comparisons post hoc test. (F) Clonal lines of edited primary patient-derived fibroblasts show high variability in mitochondrial heteroplasmy indicating different editing efficiencies per cell (Illumina NGS). N = 17, 18 and 11 for replicates 1–3, respectively. (G) Percentage of edited mDNA in primary patient-derived fibroblasts over time. This percentage slightly increased over time in most bulk and clonal cultures (Sanger sequencing). (H) Imaging flow cytometry analysis after TMRM-staining shows improved mitochondrial membrane potential in highly edited but not in lowly edited fibroblast lines (normalized to uncorrected control fibroblasts (red dashed line)). Green dashed line: average membrane potential of healthy control (HC) fibroblast lines from three individuals. N = 3 for 35%-edited line and HC, N = 4 for 76%-edited line, N = 1 for 81%-edited line; t test between 76% edited and 35% edited lines. (I) Oxygen Consumption Rate (OCR) assessed by mitochondrial stress test on 81%-edited patient fibroblasts conditioned with either glucose or galactose, measured in basal conditions and after sequential injections of the following molecules modulating mitochondrial activity: oligomycin, FCCP, rotenone and antimycin-A. N = 5 technical replicates. (J) Basal respiration measured in 4 biological replicates. (K) ATP production in mitochondrial and glycolytic fractions measured in 4 biological replicates. N = 4; Error bars indicate Mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001; one-way ANOVA with Tukey’s multiple comparisons test. Raw data are provided in S1 Data.

Fig 3. LNP-delivered modRNA encoding DdCBE efficiently corrects m.DNA mutations.

(A) modRNA-encoded DdCBE delivery to liver organoids greatly increases transfection rates compared to DNA. N = 3 and N = 4 for DNA and modRNA-transfection, respectively. (B) modRNA-mediated DdCBE editing is more efficient than DNA-mediated editing to correct the m.15150G > A mutation in organoids as measured by amplicon sequencing (Illumina NGS). N = 4. (C) Cell viability as measured by propidium iodide (PI) positivity shows increased cell death for organoids electroporated with DNA but not for modRNA. N = 3. (D) modRNA-encoded DdCBE delivery to fibroblasts greatly increases transfection rates compared to DNA. N = 3. (E) Editing efficiency (Illumina NGS) of the m.4291T > C mutation six days after transfection with modRNA. N = 4. (F) Overview of LNP production. (G) Percentage of GFP-positive cells three days after LNP transfection. N = 3. (H) Efficiency of correction of m.4291T > C in patient fibroblasts (Sanger sequencing) using modRNA-DdCBE delivered with LNPs. N = 3. All P-values were calculated with Student t test, or ANOVA (C). Raw data are provided in S1 Data.

Fig 4. Off-target editing analysis in m.4291C-corrected fibroblasts.

(A) Heatmap of C•G > T•A substitution abundances (percentage of reads) within spacing regions at nine different off-target sites with homology to the m.4291-L2- and m.4291-R2-TALE target sequences in 78%-edited patient fibroblasts. (B) Heatmap of C•G > T•A substitution abundances for the most abundant substituted cytosines (highlighted in A) within the nine spacing regions in three different gene-corrected fibroblasts and uncorrected L2-only control fibroblasts. Replicates 1–3 had 78%, 20% and 46% on-target editing, respectively. (C) C•G > T•A substitution abundances as fraction of reads across the mitochondrial genome for all six fibroblast lines. The on target editing at m.4291 is depicted as T > C substitution and therefore lower in L2R2-conditions. The presence of homoplasmic substitutions in all six lines indicates naturally occurring variants. Replicate 1 contains in both L2-only and L2R2 a heteroplasmic m.1428G > A variant. Data in (A) and (B) produced with Illumina NGS, data in (C) produced with Illumina NovaSeq. Raw data are provided in S1 Data.