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Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy


Duchenne muscular dystrophy (DMD) is a monogenic disorder and a candidate for therapeutic genome editing. There have been several recent reports of genome editing in preclinical models of Duchenne muscular dystrophy1,2,3,4,5,6, however, the long-term persistence and safety of these genome editing approaches have not been addressed. Here we show that genome editing and dystrophin protein restoration is sustained in the mdx mouse model of Duchenne muscular dystrophy for 1 year after a single intravenous administration of an adeno-associated virus that encodes CRISPR (AAV-CRISPR). We also show that AAV-CRISPR is immunogenic when administered to adult mice7; however, humoral and cellular immune responses can be avoided by treating neonatal mice. Additionally, we describe unintended genome and transcript alterations induced by AAV-CRISPR that should be considered for the development of AAV-CRISPR as a therapeutic approach. This study shows the potential of AAV-CRISPR for permanent genome corrections and highlights aspects of host response and alternative genome editing outcomes that require further study.

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Fig. 1: Genome editing is sustained for 1 year in neonatal mice treated by intravenous administration.
Fig. 2: Host response to AAV-CRISPR for DMD.
Fig. 3: In vivo genome editing generates diverse on-target genome modifications including AAV integrations and aberrant splicing.
Fig. 4: AAV integrations into the Dmd locus and genome-wide.

Data availability

All custom code for reproducing Figs. 1c,d, 3b,d, and 4a–d have been made available online ( All sequencing data used in this study have been deposited in the National Center for Biotechnology Information Sequence Read Archive (SRA) database (SRP157083). Full uncropped gels are included as Source data. All other relevant raw data are available from the corresponding author on request.


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This work has been supported by Sarepta Therapeutics, the Allen Distinguished Investigator Program through The Paul G. Allen Frontiers Group, the Muscular Dystrophy Association (grant MDA277360), a Duke–Coulter Translational Partnership Grant, a Duke/UNC-Chapel Hill CTSA Consortium Collaborative Translational Research Award, NIH grant R01AR069085, an NIH Director’s New Innovator Award (DP2OD008586) and the Office of the Assistant Secretary of Defense for Health Affairs, through the Duchenne Muscular Dystrophy Research Program under awards W81XWH-15-1-0469 and W81XWH-16-1-0221. C.E.N. was supported by a Hartwell Foundation Postdoctoral Fellowship and the NIH Pathway to Independence Award (K99EB023979). J.N.R.-H. was supported by a National Science Foundation Graduate Research Fellowship and American Heart Association Predoctoral Fellowship (17PRE33350013).

Author information

Authors and Affiliations



C.E.N. designed and conducted experiments, Y.W. provided expertise on assays to measure immune responses including ELISpot, M.P.G. conducted experiments and mouse procedures, M.L.O. purified proteins and performed histological analyses, M.A.W. completed qPCR experiments, J.D.B. performed histological analyses, J.N.R.-H. performed creatine kinase assays, K.B. performed mouse procedures, R.M.C.R. produced recombinant AAV, A.A., J.H.C. and C.A.G. designed experiments, C.E.N. and C.A.G. wrote and revised the manuscript.

Corresponding author

Correspondence to Charles A. Gersbach.

Ethics declarations

Competing interests

J.N.R.-H., C.E.N. and C.A.G. have filed patent applications related to genome editing for Duchenne muscular dystrophy. C.A.G. is an advisor for Sarepta Therapeutics, and a co-founder of and advisor for Element Genomics and Locus Biosciences. A.A. is a co-founder of and advisor for StrideBio.

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

Extended data

Extended Data Fig. 1 Illumina Nextera-based unidirectional sequencing shows diverse genome changes including deletions, AAV integration, inversions and indel formation.

a, Total editing for local administration. b, Total editing for systemic administration in neonates. c,d, Deletion frequency. e,f, Inversion frequency. g,h, AAV integration frequency. i,j, Indel frequency. Data are mean ± s.e.m. Locally injected mice, n = 6; systemically injected mice, n = 4.

Extended Data Fig. 2 On-target genome editing activity in somatic and germline tissues.

a, Deletion PCR across the dystrophin locus shows Cas9 activity in multiple somatic tissues, including the liver, spleen, lung, pancreas and brain, which all show evidence of targeted gene deletion. There is no detectable deletion in the kidney samples. Cas9 expression is driven by a constitutive CMV promoter. This result is consistent with AAV8 tissue tropism45. The off-target tissue-editing experiment was conducted once. b, Deletion PCR of genomic DNA from the testis is mostly negative and undetectable in sperm. AAV integration was only detected in one testis sample and no sperm samples. The germline experiment was conducted once. c, Deep sequencing of testis DNA indicates low levels of indel formation for both AAV8- and AAV9-injected mice that were injected as neonates. Data are mean ± s.e.m. n = 4 mice in all treated groups; n = 5 untreated mice.

Extended Data Fig. 3 Complete panel of histology shows a decrease in dystrophin staining following local administration and an increase in dystrophin at the 1-year time point in systemically treated mice.

a, The histology images indicate a reduction in dystrophin after local injections, consistent with genomic and transcript data. b, Systemic samples show increased dystrophin expression after 1 year. Increased background at the 1-year time point may be a result of fibrosis in the tissue at the later time point. Representative images shown in Fig. 1 are highlighted in red. Scale bars, 200 µm. Dystrophin-restoration experiments were conducted once for each treatment group.

Extended Data Fig. 4 AAV delivery of SaCas9 elicits humoral and cellular immune responses.

a, Mice were administered with an AAV carrying a deactivated, nuclease-null SaCas9 transcriptional repressor (dSaCas9-KRAB) from a related study23. Serum was collected at 4, 8, 16 and 26 weeks. The IgG response invariably developed by 8 weeks in all tested mice and continued to increase until the 16-week time point. Data are mean ± s.e.m. (n = 4 individual mice). The dotted line indicates the end of the linear range of the standard. b, ELISpot shows T cell responses in treated adults but not neonates regardless of administration route. Spot-forming cells (SFCs) were detected in mice injected with AAV9-SaCas9 in adults at 2 weeks and 8 weeks after systemic administration, as well as 8 weeks after intramuscular injection. SFCs were not detected in mice treated as neonates. c, Complete image panel of ELISpot data. Two separate plates are shown. Data are mean ± s.e.m. n = 3 individual mice, biological replicates (BR) were used with two technical replicates (TR) as individual isolates from each mouse. d, Mice administered locally show increased IFNγ and reduced FOXP3 and IL-12β expression, whereas mice administered systemically as adults or neonates show no significant changes. Statistics calculated compared to untreated, t-test with Holm–Bonferroni multiple comparisons correction was used. n = 4, IM-AAV8; n = 3, IV-AAV8; n = 4, Neonate-AAV8.

Extended Data Fig. 5 Complete quantitative and reproducibility data for the Illumina Nextera-based unidirectional sequencing measures.

Data associated with Fig. 3b. a, Quantitative data for genome-editing measurements are an average of n = 4 individual mice. Skeletal and cardiac muscle are shown on a separate scale from liver samples. b, Comparison of deep sequencing for both gRNAs. cg, Comparison of indel rates for gRNA1 to identify alternate modifications. hl, Comparison of indel rates of gRNA2 with alternate modifications. Rare events have poorer correlations. An estimated limit of detection is given based on the inversions detected that range between 0.1% and 0.2%. The limit of detection could be decreased with more input DNA and increased number of reads to detect more rare events, possibly including translocations.

Extended Data Fig. 6 Deep sequencing of target loci for gRNA1 and gRNA2 in multiple tissues and treatment routes show indel formation and short AAV insertions.

a, Mice treated as 8-week-old adults by injection into the tibialis anterior were euthanized and tissues were collected at 8 weeks and 6 months after a single administration. b, Systemic administration in neonates by FVI of AAV8 or AAV9 was followed by analysis at 8 weeks and 1 year. c, Systemic administration in adults by tail-vein injection was followed by tissue collection at 12 weeks after the administration. d, Local administration with AAV8 (n = 6, n = 5, two-tailed t-test). e, Systemic administration with AAV8 in neonates (n = 4, two-way ANOVA). f, Systemic administration in neonates with AAV9 (n = 4). g, Tail-vein administration in adults with AAV8 (n = 3). h, Tail-vein administration in adults with AAV9 (n = 3). im, The same administration at the gRNA2 loci. n, Small AAV insertions were detected by deep sequencing for insertions that range from 10 to 45 bp in length. These insertions account for a small subset of integrations detected by Nextera-based sequencing. Nextera-based sequencing shows a higher detection rate of AAV genome insertions (n = 8). o, Short AAV insertions detected by indel sequencing are almost exclusively located within the ITR regions of the AAV vector genome. Data are mean ± s.e.m.

Extended Data Fig. 7 Illumina Nextera-based unidirectional sequencing of cDNA shows transcript changes over time in systemically administered neonates.

There are notable differences in the number of circular RNA events and multi-skipping events when sequencing from the forward or reverse direction, which indicates that alternative splicing may be preferred in reverse direction. a-b) intact unedited transcripts, c-d) exon 23 deleted transcripts, e-f) AAV-fusion transcripts, g-h) transcripts with multiple skipped exons, i-j) circular transcripts, k-l) transcripts with alternative splicing, m-n) and transcripts with intron inclusion. Data are mean ± s.e.m. (n = 4 for all samples).

Extended Data Fig. 8 Nextera-based sequencing reveals dystrophin-AAV transcript fusions and is a reproducible method.

a, Web logo map of nucleotide preference for spicing of dystrophin transcript to AAV vector genome shows canonical splicing is preferred. The forward read strategy priming from exon 22 shows that dystrophin-AAV splice fusions prefer an AG as the canonical splice acceptor. The sequencing read is shown as a black box. b, Similarly, the reverse priming strategy shows the preference for the canonical GT splice donor before the AAV-dystrophin fusion. The dotted-line box is not in the sequencing read so the AG or GT are revealed by alignment with the vector genome. Web logo maps were generated with the online tool46 at c, Deletions as measured by the Nextera method show a higher estimation by the reverse-sequencing method than the forward-sequencing method. d,e, ddPCR measures higher levels of gene deletion than either Nextera-based strategy. All comparisons were consistent (R2 > 0.99). The right Nextera-based method had an order of magnitude higher read count and there is potential bias for transposon recombination. y = x is plotted as a blue dotted line. Data are mean ± s.e.m., all data are from n = 4 mice.

Extended Data Fig. 9 Optimization of the Nextera-based sequencing method.

a, Multiple conditions were tested to find an optimized protocol. The standard method from the manufacturer’s protocol is shown as ‘S’ and the optimized condition that we identified as ‘O’. b, To test whether the random tagmentation works, a nested PCR was performed after the first PCR. The nested PCR used primer-binding sites of known amplicon size to detect the presence of expected products, including the unmodified target locus and the intended deletion. Only the optimized condition revealed all four predicted amplicons (5′ and 3′ enrichment for both products). We suspect that the standard condition generates fragments that are too short and lose the test-primer-binding site. By contrast, the optimized protocol generates longer DNA fragments that maintain the primer-binding site and would be better suited for unbiased sequencing. Optimization was performed only once. c, Gel showing amplicons after the first PCR. d, Gel showing amplicons after adding barcodes and adapters. Bands were purified within the outlined box. Gel images are representative of each sample analysed by deep sequencing (n = 3 independent experiments). e, Each method shows varying quantities of reads aligned to the reference because of mispriming. gDNA reads show that the gRNA1–intron22 strategy had a higher percentage of reads aligned to the genome (n = 7). f, The cDNA method shows a higher percentage of aligned reads for the exon 24 reverse priming strategy (n = 7). g, Sequencing out of the AAV shows 6.26% of reads aligned to the reference with the majority of aligned reads consisting entirely of AAV vector episomes with no novel junctions, represented in black. In blue are reads that did not align to the mouse reference genome or were within repetitive regions that made identification impossible. Green labels indicate the 0.04% of reads that aligned to unique loci within the mouse genome. These reads are listed in detail in Supplemental Table 1, the majority of the reads are in the targeted Dmd locus. Data are mean ± s.e.m.

Supplementary information

Source data

Source Data Fig.1

Unprocessed histological images and western blots

Source Data Methods

Western blots for preparation of recombinant SaCas9

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Nelson, C.E., Wu, Y., Gemberling, M.P. et al. Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy. Nat Med 25, 427–432 (2019).

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