In vivo base editing rescues Hutchinson–Gilford progeria syndrome in mice

Abstract

Hutchinson–Gilford progeria syndrome (HGPS or progeria) is typically caused by a dominant-negative C•G-to-T•A mutation (c.1824 C>T; p.G608G) in LMNA, the gene that encodes nuclear lamin A. This mutation causes RNA mis-splicing that produces progerin, a toxic protein that induces rapid ageing and shortens the lifespan of children with progeria to approximately 14 years1,2,3,4. Adenine base editors (ABEs) convert targeted A•T base pairs to G•C base pairs with minimal by-products and without requiring double-strand DNA breaks or donor DNA templates5,6. Here we describe the use of an ABE to directly correct the pathogenic HGPS mutation in cultured fibroblasts derived from children with progeria and in a mouse model of HGPS. Lentiviral delivery of the ABE to fibroblasts from children with HGPS resulted in 87–91% correction of the pathogenic allele, mitigation of RNA mis-splicing, reduced levels of progerin and correction of nuclear abnormalities. Unbiased off-target DNA and RNA editing analysis did not detect off-target editing in treated patient-derived fibroblasts. In transgenic mice that are homozygous for the human LMNA c.1824 C>T allele, a single retro-orbital injection of adeno-associated virus 9 (AAV9) encoding the ABE resulted in substantial, durable correction of the pathogenic mutation (around 20–60% across various organs six months after injection), restoration of normal RNA splicing and reduction of progerin protein levels. In vivo base editing rescued the vascular pathology of the mice, preserving vascular smooth muscle cell counts and preventing adventitial fibrosis. A single injection of ABE-expressing AAV9 at postnatal day 14 improved vitality and greatly extended the median lifespan of the mice from 215 to 510 days. These findings demonstrate the potential of in vivo base editing as a possible treatment for HGPS and other genetic diseases by directly correcting their root cause.

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Fig. 1: ABE-mediated correction of the LMNA c.1824 C>T mutation in fibroblasts derived from patients with progeria.
Fig. 2: Analysis of off-target DNA and RNA editing and gene expression changes after treating fibroblasts derived from patients with progeria with ABEmax-VRQR.
Fig. 3: DNA, RNA and protein levels after a single in vivo injection of ABE-expressing AAV9 in a mouse model of human progeria.
Fig. 4: Aortic histopathology and lifespan of progeria mice after a single injection of ABE-AAV9.

Data availability

DNA-sequencing files can be accessed at the NCBI SRA with accession code PRJNA627465. Raw data are available in Supplementary Data 1, 2, 3, 5 and 6.

Code availability

Code used in this study is available from https://github.com/CwilsonBroad/Koblan_2020_In-Vivo-Adenine-Base-Editing-Corrects-Hutchinson-Gilford-Progeria-Syndrome (Supplementary Note 3).

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Acknowledgements

This work was supported by NIH grants U01AI142756, UG3AI150551, UG3TR002636, RM1HG009490, R01EB022376, R35GM118062, Z01HG200305, R01HL146654 and R01HL126784, and by the Howard Hughes Medical Institute. L.W.K. acknowledges an NSF GRFP fellowship; C.W. acknowledges a Damon Runyon Cancer Research Foundation fellowship (DRG-2343-18); G.A.N. acknowledges a Helen Hay Whitney postdoctoral fellowship; and L.B.G., L.W.K. and D.R.L. acknowledge support from The Progeria Research Foundation.

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Authors

Contributions

L.W.K., J.D.B., C.Y.L., M.R.E., F.S.C. and D.R.L. designed the research. L.W.K., M.R.E., C.W., W.A.C., L.D., Y.G.G., X.M., G.A.N., S.P.D., J.D.B. performed cell culture experiments. L.W.K., M.R.E., C.W., W.A.C., J.M.L., Z.X., U.L.T., L.D., S.P.D., N.N., Q.S., C.K. and J.D.B. performed in vivo experiments. L.B.G., K.C., F.S.C., J.D.B. and D.R.L. supervised the project. L.W.K. and D.R.L. wrote the manuscript with input from all other authors.

Corresponding authors

Correspondence to Francis S. Collins or Jonathan D. Brown or David R. Liu.

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Competing interests

D.R.L. is a co-founder of Beam Therapeutics, Prime Medicine, Pairwise Plants and Editas Medicine, companies that use genome editing. The remaining authors declare no competing interests.

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Peer review information Nature thanks the anonymous reviewers for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Additional characterization of patient-derived cells treated with ABE7.10max-VRQR.

a, Bystander V690A editing in patient-derived HGADFN167 and HGADFN188 cells 20 days after treatment with lentiviral ABE7.10max-VRQR. b, Indel formation frequency at the c.1824 target locus in HGADFN167 and HGADFN188 cells 20 days after treatment with lentiviral ABE7.10max-VRQR. Data are mean ± s.d. for n = 5 technical replicates (individual points) for a, b. c, Sanger DNA sequencing traces of untreated HGADFN167 cells, 20-day treated HGADFN167 cells and unaffected control cells. The target nucleotide is boxed. d, qPCR-normalized progerin mRNA abundance in cells described in c. Data are mean ± s.d. for n = 3 biological replicates. e, Western blot analysis of HGADFN167 cells described in c. LMNA, progerin, and LMNC protein are all stained on the gel, A GAPDH loading control is shown below. An additional replicate is provided in Fig. 1d. Unaff. ctrl, control cells from an unaffected parent. f, Sanger DNA sequencing traces of untreated, non-targeting (NT)-sgRNA-treated and ABE-treated HGADFN155 fibroblasts at a 20-day time point to ensure NT-sgRNA did not lead to DNA editing. g, Western blot analysis of cells described in f as well as treated cells at a 30-day time point. LMNA, progerin, and LMNC proteins are all stained on the gel, a β-actin loading control is shown below. Expected molecular weights: lamin A, 74 kDa; progerin, 69 kDa; lamin C, 65 kDa. Complete blots are available in Supplementary Fig. 1. Additional replication was not performed. h, HGADFN167 (left) and HGADFN188 (right) cell lines untreated or treated with lentiviral ABE7.10max-VRQR after 10 or 20 days show similar relative distributions of A-to-I SNVs in their transcriptomes compared with the hg38 human genome reference sequence. On average, 36 ± 3.6% of SNPs in these samples occur with around 100% frequency, suggesting they arise from genomic sequence variations; however, we cannot explicitly exclude them from consideration as no whole-genome sequence is available for these cell lines. Raw counts of 100% edited SNPs per sample are: untreated HGADFN167 cells (849), HGADFN167 10 d after treatment (883), HGADFN167 20 d after treatment (871), untreated HGADFN188 cells (488), HGADFN188 10 d after treatment (501), HGADFN188 20 d after treatment (510).

Extended Data Fig. 2 CIRCLE-seq analysis of HGADFN167 and HGADFN188 cells using Cas9-VRQR and the progeria-targeting sgRNA.

a, b, CIRCLE-seq read counts for Cas9-VRQR nuclease-treated genomic DNA from HGADFN167 (a) and HGADFN188 (b) cell lines. Targeted amplicon sequencing was used to assess the off-target base editing for 36 noted total loci distributed across both cell lines. DNA at 32 of 35 loci amplified efficiently from both cell lines (denoted by black check marks), DNA at 3 loci failed to amplify (denoted by red X marks). Complete CIRCLE-seq data are provided in Supplementary Data 4. c, SDS–PAGE gel stained with InstantBlue to follow protein purification of Cas9-VRQR. Totals of 0.5 μl clarified lysate, 0.25 μl nickel column elution or 0.1 μl of the concentrated protein stock after His-tag purification and ion-exchange chromatography were added to 5 μl NuPAGE loading buffer. Samples were denatured at 98 °C for 5 min before loading onto the 4–12% acrylamide gel. A Precision Plus Protein Kaleidoscope Pre-Stained Ladder (Bio-Rad) was used as reference. The desired Cas9-VRQR has a predicted molecular weight of 161.9 kDa. Additional replication was not performed.

Extended Data Fig. 3 DNA on-target editing, bystander editing and indel efficiencies across tissues from in vivo injection route optimization experiments.

a, Dual AAV9 encoding split-intein ABE7.10max-VRQR base editor halves and the LMNA-targeting sgRNA were injected into homozygous LMNA c.1824 C>T mice. P3 retro-orbital (RO) injections (5 × 1010 vg of each AAV, 1 × 1011 vg total), P14 RO injections (5 × 1011 vg of each AAV, 1 × 1012 vg total) and P14 intraperitoneal (IP) injections (5 × 1011 vg of each AAV, 1 × 1012 vg total) were tested. At 6-weeks of age, mice were euthanized and heart, muscle, liver, aorta and bone were isolated for sequencing analysis. Tissues were sub-sectioned for sequencing analysis to ensure sub-sections did not show differences in editing efficiencies for downstream analyses. Each bar represents a different tissue subsection. DNA-editing efficiencies correcting LMNA c.1824 from T (pathogenic) to C (wild-type) for P3 RO-injected mice (left, n = 4), P14 RO-injected mice (middle, n = 5) and P14 IP-injected mice (right, n = 5) at 6 weeks of age are shown for five disease-relevant tissues. Data are mean ± s.d. b, Apparent LMNA c.1824 T (pathogenic) to C (wild-type) mutations from tissue samples of saline-injected P3 RO (left) and P14 RO (right) control mice at 6 months of age show background signal due to amplicon crossover during PCR between the human diseased allele and the wild-type mouse allele, which share 90% overall sequence identity within the amplified region. Similar crossover levels were observed across 11 tissues in both P3 RO and P14 RO saline-injected mice. Data are mean ± s.d. for n = 12 mice (6 male, 6 female). c, Computational filtering of same sequencing reads shown in b after removing any reads containing any mouse-specific sequence variations, analysing only reads containing exclusively human sequence. The script used to remove mouse-containing sequencing reads is in the link in Supplementary Note 3 and is described in the Methods (‘High-throughput sequencing of in vivo samples’). d, DNA editing for P3- and P14-injected mice at 6 months of age across 11 tissues. Each point represents a biological replicate of a tissue taken from a unique mouse (n = 12 for each group). e, V690A bystander editing frequency across eleven tissues for P3 RO and P14 RO ABE-treated mice at 6 months of age (n = 12 for each group). f, Indel frequencies at the c.1824 target locus across 11 tissues for P3 RO and P14 RO ABE-treated mice at 6 months of age. Data are mean ± s.d. for the indicated number of biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s unpaired two-sided t-test.

Extended Data Fig. 4 Quantification of LMNA and progerin transcript abundance by ddPCR in mice that were retro-orbitally injected with saline or ABE-AAV9 at P3 or P14.

a, ddPCR counts for LMNA (grey bars) and progerin (red bars) RNA transcript abundance in P3 RO saline- and ABE-AAV9-injected mice. Data are mean ± s.d. for n = 12 mice. b, ddPCR counts for LMNA (grey bars) and progerin (red bars) RNA transcript abundance in P14 RO saline- and ABE-AAV9-injected mice. Data are mean ± s.d. for n = 12 biological replicates for all samples except for saline-injected mouse skin (n = 11), WAT (n = 7), visceral fat (n = 11), tibia (n = 11) and aorta (n = 8); and ABE-AAV9-injected mouse WAT (n = 11), tibia (n = 9) and aorta (n = 10). Visc. fat, visceral fat. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s unpaired two-sided t-test for a, b. Liver and heart values are reproduced from Fig. 3c for ease of comparison.

Extended Data Fig. 5 Quantification of western blots.

Liver, heart and aorta tissue western blots for P3-injected (top half of each tissue set) and P14-injected mice (bottom half of each tissue set) were quantified by western blot. Samples from females appear in the left column, and samples from males are in the right column. Each lane represents the tissue type specified on the left taken from a different mouse. Control mice were treated with saline instead of ABE-AAV9. WT indicates C57BL/6 mouse lacking the transgene, showing that the antibody is specific to human lamin proteins and progerin. The abundance of lamin A or progerin protein relative to β-actin in saline- or ABE-treated mouse tissues was quantified by normalizing the fluorescence signal from the secondary antibody for each band (800 nm for progerin and lamins, and 680 nm for actin; see Methods (‘Protein isolation and western blotting from mouse tissues’). The normalized protein abundance relative to saline-treated samples (set to 100) is shown in the bar graphs. Control mice were treated identically to the corresponding ABE-treated mice except injected with saline instead of ABE-AAV9. Raw fluorescent signal for progerin protein measured at the 800-nm wavelength (using IRDye-labelled antibody) is displayed under each lane. Data are mean ± s.d. for n = 5 or 6 biological replicates, as indicated. The n for each sample type is listed in each figure panel. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s unpaired two-sided t-test. Expected molecular weights: lamin A, 74 kDa; progerin, 69 kDa; lamin C, 65 kDa. Liver and heart blots are reproduced from Fig. 3c for ease of comparison. Complete blots are available in Supplementary Fig. 1.

Extended Data Fig. 6 Aortic histology of male and female mice that were retro-orbitally injected with saline or ABE-AAV9 at P3.

a, Representative aorta cross-sections for P3 RO saline- or ABE-AAV9-injected males at 6 months of age. Left images were stained with H&E; right images were stained with Movat’s pentachrome stain. b, Representative aorta cross-sections for P3 RO saline- or ABE-AAV9-treated females at 6 months of age. Left images were stained with H&E; right images were stained with Movat’s pentachrome stain. Unaffected WT are wild-type C57BL/6 mice. WT M2, 9609M, 9177M, WT F3, 9628F and 9148F are reproduced from Fig. 4 for ease of comparison. These sections each represent replicates from different mice.

Extended Data Fig. 7 Aortic histology of male and female mice that were retro-orbitally injected with saline or ABE-AAV9 at P14.

a, Representative aorta cross-sections for P14 RO saline- or ABE-AAV9-injected males at 6 months of age. Left images were stained with H&E; right images were stained with Movat’s pentachrome stain. b, Representative aorta cross-sections for P14 RO saline- or ABE-AAV9-treated females at 6 months of age. Left images were stained with H&E; right images were stained with Movat’s pentachrome stain. Unaffected WT are wild-type C57BL/6 mice, reproduced from Extended Data Fig. 9a, b for ease of comparison. Images from the following mice are reproduced from Fig. 4 for ease of comparison: WT M2, 9440M, 9459M, WT F3, 9536F and 9535F. These sections each represent replicates from different mice.

Extended Data Fig. 8 Immunofluorescence staining of aortas from male and female control C57BL/6 mice, untreated mice and mice that were retro-orbitally injected with saline or ABE-AAV9 at P14.

Immunofluorescence staining of C57BL/6 (n = 2), untreated P28 homozygous LMNA c.1824 C>T (n = 1), saline-treated homozygous LMNA c.1824 C>T (n = 4) and ABE-treated homozygous LMNA c.1824 C>T (n = 5) mouse aortas stained for human lamin A/C + DAPI or for progerin + DAPI. Scale bars, 10 μm. Images from untreated 28 day-old, WT M1, 9424M and 9464M are replicated from Fig. 4 for ease of comparison.

Extended Data Fig. 9 Skin histology of male and female mice that were retro-orbitally injected with saline or ABE-AAV9 at P3.

a, Representative skin cross-sections for P3 RO saline-injected (left), ABE-AAV9-injected (middle) and wild-type C57BL/6 males at 6 months of age. Left images were stained with H&E; right images were prepared with Masson’s trichrome staining. b, Representative skin cross-sections for P3 RO saline-injected (left), ABE-AAV9-injected (middle) and wild-type C57BL/6 females at 6 months of age. Left images were stained with H&E; right images were prepared with Masson’s trichrome staining. Unaffected WT are wild-type C57BL/6 mice. These sections each represent replicates from different mice.

Extended Data Fig. 10 Skin histology of male and female mice that were retro-orbitally injected with saline or ABE-AAV9 at P14.

a, Representative skin cross-sections for P14 RO saline-injected (left), ABE-AAV9-treated (middle) and wild-type C57BL/6 males at 6 months of age. Left images were stained with H&E; right images were prepared with Masson’s trichrome staining. b, Representative skin cross-sections for P14 RO saline-injected (left), ABE-AAV9-treated (middle) and wild-type C57BL/6 females at 6 months of age. Left images were stained with H&E; right images were prepared with Masson’s trichrome staining. Unaffected WT are wild-type C57BL/6 mice, reproduced from Extended Data Fig. 12a, b for ease of comparison. These sections each represent replicates from different mice.

Extended Data Fig. 11 Body weights of mice that were retro-orbitally injected with saline or ABE-AAV9 at P3 or P14.

Weights of homozygous LMNA c.1824 C>T mice taken across mouse lifespans for cohorts of P3 RO (left) and P14 RO (right) saline- and ABE-AAV9-injected cohorts. Mouse weights are shown by sex. The x axis shows days post-injection, rather than age. Data are mean ± s.d. for the number of surviving mice at each time point; complete data can be accessed in Supplementary Data 5.

Extended Data Fig. 12 Whole-genome sequencing analysis of SNVs and indels and quantification of RAB25 transcript levels in mouse tissue samples.

a, Distribution of all possible SNV types in non-tumour liver tissue and liver tumour tissue samples isolated from ABE-AAV9-injected and saline-injected mice. Values from individual tissue samples are shown on the left. Aggregated values from all AAV-injected mouse tumour tissue samples, all AAV-injected mouse liver tissue samples and all saline-injected mouse liver tissue samples are shown on the right. Data are mean ± s.d. with each tissue section treated as a different sample: AAV-injected tumour tissue (n = 7), AAV-injected liver tissue (n = 6) and saline-injected liver tissue (n = 2). b, Genomic classification of A•T-to-G•C SNVs. Values from individual tissue samples are shown on the left. Aggregated values from AAV-injected mouse tumour tissue samples, AAV-injected mouse liver tissue samples and saline-injected mouse liver tissue samples from all tissue types are shown on the right. Data are mean ± s.d. with each tissue section treated as a different sample: AAV-injected tumour tissue (n = 7), AAV-injected liver tissue (n = 6), saline-injected liver tissue (n = 2). c, A•T-to-G•C SNVs and indels found in or near genes that are recurrently mutated in human liver cancers63,64 including introns, exons, and at ATAC-seq-defined cis-regulatory regions within 100-kb of each gene’s transcription start site, in AAV-injected mouse tumour tissue samples, AAV-injected mouse liver tissue samples and saline-injected mouse liver tissue samples. Data are mean ± s.d. Individual data points are shown for each sample. The complete list of SNVs from ANNOVAR analysis is provided as Supplementary Data 6. Summary statistics for SNV calls are in Supplementary Data 2. d, RNA isolated from mouse liver tissue samples was reverse-transcribed and amplified with primer sets specific to mouse Gapdh (detected with Cy5), mouse Actb (detected with Cy5.5) and human RAB25 (detected with TEX 615). Ct values were determined by quantitative PCR and are shown below each lane. N.D., not detected.

Supplementary information

Supplementary Information

This file contains Supplementary Notes 1-3, Supplementary Figure 1, Supplementary Tables 1-2 and Supplementary References.

Reporting Summary

Supplementary Data

Supplementary Data 1: AAV integration analysis from mouse whole-genome sequencing data.

Supplementary Data

Supplementary Data 2: Analysis of SNVs from mouse whole-genome sequencing data.

Supplementary Data

Supplementary Data 3: Liver cancer-associated SNVs and indels in mouse wholegenome sequencing data.

Supplementary Data

Supplementary Data 4: CIRCLE-seq candidate off-target loci genomic coordinates and amplification primers.

Supplementary Data

Supplementary Data 5: Raw data for in vivo samples.

Supplementary Data

Supplementary Data 6: All SNVs from mouse whole-genome sequencing data.

Video 1

Representative untreated male progeria mouse (not enrolled in this study), taken at 7.5 months of age.

Video 2

Representative male progeria mice injected at P14 with ABE-AAV, taken at 9.75 months of age.

Video 3

Representative female progeria mice injected at P14 with ABE-AAV, and wild-type C57BL/6 female mouse, taken at 9.75 months of age.

Video 4

Representative male progeria mice injected at P14 with ABE-AAV, and wild-type C57BL/6 male mouse, taken at 11 months of age.

Video 5

Representative female progeria mice injected at P14 with ABE-AAV, taken at 11 months of age.

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Koblan, L.W., Erdos, M.R., Wilson, C. et al. In vivo base editing rescues Hutchinson–Gilford progeria syndrome in mice. Nature (2021). https://doi.org/10.1038/s41586-020-03086-7

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