Chemical synthesis of 3-((4-(Dimethylamino)butanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy) methyl) propyl (9Z)-tetradec-9-enoate (TCL053)
To a mixture of 2,2-bis(hydroxymethyl)propane-1,3-diol (5.45 g), 1H-imidazole (2.72 g) and DMF (190 mL), a solution of tert-butylchlorodimethylsilane (3.01 g) in DMF (10 mL) was added at room temperature. After stirring for 24 h, the reaction mixture was concentrated under reduced pressure. The residue was diluted with ethyl acetate, washed three times with water and once with saturated brine, and then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/hexane) to afford the 2-(((tert-Butyldimethylsilyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diol compound (2.25 g).
1H NMR (300 MHz, CDCl3) δ ppm 0.08 (6H, s), 0.90 (9H, s), 2.53 (3H, t, J = 5.5 Hz), 3.66 (2H, s), 3.73 (6H, d, J = 5.5 Hz)
Next, to a solution of 2-(((tert-butyldimethylsilyl)oxy)methyl)-2-(hydroxymethyl) propane-1,3-diol (258 mg), (9Z)-tetradec-9-enoic acid (769 mg) and DMAP (126 mg) in DMF (3 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (790 mg) was added at room temperature. After stirring for 18 h, the reaction mixture was diluted with ethyl acetate, washed twice with water and once with saturated brine, and then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (NH, ethyl acetate / hexane) to afford the 3-((tert-Butyl(dimethyl)silyl)oxy)−2,2-bis(((9Z)-tetradec-9-enoyloxy)methyl) propyl (9Z)-tetradec-9-enoate compound (860 mg).
1H NMR (300 MHz, CDCl3) δ ppm 0.03 (6H, s), 0.81-0.96 (18H, m), 1.18-1.41 (36H, m), 1.53-1.67 (6H,m), 1.91-2.10 (12H, m), 2.29 (6H, t, J = 7.6 Hz), 3.58 (2H, s), 4.08 (6H, s), 5.27-5.43 (6H, m)
Third, to a solution of 3-((tert-butyl(dimethyl)silyl)oxy)−2,2-bis(((9Z)-tetradec-9-enoyloxy) methyl)propyl (9Z)-tetradec-9-enoate (5.91 g) in THF (120 mL), a mixture of a THF solution of TBAF (1 M, 14.85 mL) and acetic acid (4.91 mL) was added at room temperature. After stirring for 3 days, the reaction mixture was concentrated under reduced pressure. The residue was diluted with ethyl acetate, washed once with saturated aqueous solution of sodium hydrogen carbonate and once with saturated brine, and then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/hexane) to afford the 3-Hydroxy-2,2-bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl(9Z)-tetradec-9-enoate compound (4.96 g).
1H NMR (300 MHz, CDCl3) δ ppm 0.82–0.97 (9H, m), 1.16–1.42 (36H, m), 1.52–1.68 (6H, m), 1.90–2.12 (12H, m), 2.32 (6H, t, J = 7.6 Hz), 2.52 (1H, t, J = 7.0 Hz), 3.49 (2H, d, J = 7.0 Hz), 4.11 (6H, s), 5.26-5.42 (6H, m)
Finally, to a solution of 3-hydroxy-2,2-bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl (9Z)-tetradec-9-enoate (4.96 g), DMAP (796 mg) and 4-(dimethylamino)butanoic acid hydrochloride (2.19 g) in DMF (20 mL), 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (2.50 g) was added at room temperature. After stirring for 18 h, the reaction mixture was diluted with ethyl acetate, washed once with saturated aqueous solution of sodium hydrogen carbonate and once with saturated brine, and then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (NH, ethyl acetate/hexane) to afford the 3-((4-(Dimethylamino)butanoyl)oxy)−2,2-bis(((9Z)-tetradec-9-enoyloxy) methyl) propyl (9Z)-tetradec-9-enoate (TCL053) compound (5.31 g).
1H NMR (300 MHz, CDCl3) δ ppm 0.82-0.94 (9H, m), 1.20-1.42 (36H, m), 1.50-1.66 (6H, m), 1.69-1.83 (2H, m), 1.90-2.10 (12H, m), 2.20 (6H, s), 2.23-2.41 (10H, m), 4.11 (8H, s), 5.23-5.44 (6H, m), MS m/z (M + H): 874.75
LNP formulation
TCL053 and TCL065 ionizable lipids were synthesized and obtained from Drug Discovery Chemistry Labs in Takeda Pharmaceutical Company, Ltd. DLin-MC3-DMA ionizable lipid was purchased from MedChemExpress (Monmouth Junction, NJ). Structural lipids DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), and DMG-PEG (1,2-dimyristoyl-sn-glycero-3-methoxypolyethyleneglycol 2000) were purchased from NOF Corporation (Tokyo, Japan), and cholesterol from Avanti Polar Lipids (Alabaster, AL). Firefly luciferase (luc) mRNA and Cas9 mRNA (CleanCap® Cas9 mRNA (5moU), uridines were substituted with 5-methoxyuridine, catalog no. L-7206, 4521-mer) were purchased from Trilink Biotechnologies (San Diego, CA). Chemically modified sgRNAs (2’OMe and phosphorothioate modification of three nucleotides at the 5′ and 3′ end) were synthesized by GeneDesign (Osaka, Japan). Lipid components (60% ionizable lipid, 10.6% structural lipid, 27.3–28.7% cholesterol, 0.7–2.1% DMG-PEG, in mol%) were dissolved in 90% ethanol, and RNA (mRNA only, sgRNA only, or a mixture of both) was dissolved in 10 mM MES (2-(N-morpholino) ethanesulfonic acid) buffer (pH 5.5). For MC3-LNP-IV, lipid components (50% Dlin-MC3-DMA, 10% DSPC, 28.5% cholesterol, 1.5% DMG-PEG, in mol%) were dissolved in ethanol, and RNAs (Cas9 mRNA, hEx45 sgRNA #1, and hEx45 sgRNA #23) were dissolved in 50 mM sodium citrate (pH 4.0).
LNP-mRNA and LNP-sgRNA were prepared by mixing the organic lipid components and the aqueous RNA solution at a lipid:RNA ratio of 23:1 (w/w), with a microfluidic laminal flow rate of 8 mL/min (aqueous:organic flow rate ratio of 2:1) using a NanoAssemblr Benchtop (Precision Nanosystems, Vancouver, Canada). Then, the mixture was dialyzed for 1 h at 4 °C against RNase-free water followed by dialysis for 18 h at 4 °C against PBS (pH 7.4) using a Slide-A-Lyzer Dialysis Cassette 20 K MWCO (Thermo Fisher Scientific, Waltham, MA). Resultant LNP was concentrated with an Amicon Ultra 30 K MWCO (Merk Millipore, Burlington, MA) and filtrated through a 0.2-μm syringe filter (AGC Techno Glass, Shizuoka, Japan). The LNP size was determined by dynamic light scattering using a Zetasizer Nano ZS (Malvern Panalytical, Worcestershire, UK), and the RNA packaging efficiency was measured by a Quant-it RiboGreen RNA Assay Kit (Thermo Fisher Scientific), as manufacture’s instruction47.
AAV preparation
AAV2-luc was purchased from Cell Biolabs (San Diego, CA). AAV vectors containing hEx45 sgRNAs #1 and #23 were constructed by cloning synthetic oligos (Eurofins Genomics, Tokyo, Japan) into pAAV-Guide-it-Down vector (Takara Bio, Shiga, Japan). pAAV-Guide-it Up vector was purchased from Takara Bio. These vectors were packaged into AAV-DJ (Cell Biolabs) using AAVpro293 cells (Takara Bio) and purified using an AAVpro Purification Kit (Takara Bio).
Myogenic differentiation of DMD-iPS cells and LNP-CRISPR transfection
Dox-inducible MYOD1-expressing DMD patient iPS (DMD-iPS) cells (clone CiRA00111, available from the RIKEN BioResource Research Center as HPS0383) were previously generated48. The DMD-iPS cells were seeded on a Matrigel-coated plate and cultured in StemFit AK02N (Ajinomoto, Tokyo, Japan) containing 10 μM Y-27632 dihydrochloride (Tocris Bioscience, Bristol, UK). The next day, medium was changed with fresh ReproCell Primate ES Cell medium (REPROCELL, Kanagawa, Japan). After 24 h, the medium was changed to ReproCell Primate ES Cell medium supplemented with 1 μg/mL doxycycline (DOX) (Sigma-Aldrich, Saint Louis, MO). Twenty-four hours after the addition of DOX, the medium was changed to α-MEM (Sigma-Aldrich) containing 5% KnockOut Serum Replacement (Thermo Fisher Scientific) and 1 μg/mL DOX. After 3 days, LNP-Cas9 mRNA (1 μg mRNA) was transfected with LNP-hEx45 sgRNA #1 (1 μg sgRNA), LNP-hEx45 sgRNA #23 (1 μg sgRNA) or LNP-hEx45 sgRNA #1 + #23 (0.5 μg each RNA) into myoblast cells derived from DMD-iPS cells, and the cells were incubated at 37 °C for 3 days.
Generation of humanized DMD model mice and animal experiments
Human DMD exon 45 knock-in (hEx45KI) mice were generated by replacing mouse exon 45 and the flanking intronic sequence (total 1.5 kb in size) with the corresponding human exon 45 sequence by CRISPR-Cas9/sgRNA-mediated targeting, and hEx45KI-Dmd exon 44 knock-out (hEx45KI-mdx44) mice were generated by deleting mouse exon 44 with CRISPR-Cas9 dual sgRNAs (performed by Axcelead, Kanagawa, Japan). CAG-Luc2 hDMDEx45 KI mice were previously generated by targeting the Gt(ROSA)26Sor locus with a pCAGGS-Luc2-hEx45 reporter construct using the CRISPR-Cas9 system35. C57BL/6J mice were purchased from CLEA Japan (Kanagawa, Japan). All mice were housed under SPF condition with free-food and water supply with 12 h dark/light cycle, controlled temperature (around 23 °C), and controlled humidity (around 55%). Mice were anesthetized with isoflurane and intramuscularly injected with LNP, AAV or phosphorodiamidate morpholino oligomer (5′-GCTGCCCAATGCCATCCTGGAGTTCCTG-3′, obtained from Gene Tools, Philomath, OR) into the gastrocnemius (GC) or tibialis anterior (TA) muscle. We used a 29-gage (G) needle and inserted 5–6 mm to inject 50 μL (for TA, IHC analysis) or 100 μL (for GC, IVIS analysis) of the sample solution at a speed of 12–25 μL per second. For repeated injections, we tried to inject at the same site each time. For limb perfusion, a tourniquet was placed on the proximal part of the hindlimb before the injection49. LNP in a volume of 2.5–10 mL/kg was injected into the dorsal saphenous vein in approximately 10 s with a 32-G needle. Five minutes after the injection, the tourniquet was removed. All in vivo experiments were evaluated and approved by the Institutional Animal Care and Use Committee at Takeda Pharmaceutical Company, Ltd.
Detection of luciferase bioluminescence
Mice were anesthetized with isoflurane and intraperitoneally administered 3 mg D-Luciferin (Promega, Madison, WI) that was dissolved in PBS. Ten minutes after the injection of D-Luciferin, images were captured using IVIS. Hairs around the GC and abdomen of mice were shaved prior to imaging to increase the sensitivity of the luminescence detection.
Measurement of exon skipping efficiency
Total RNA of DMD-iPS cell-derived myoblast cells were extracted by RIPA buffer (FUJIFILM Wako Pure Chemical, Osaka, Japan) and an miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Extracted total RNA was reverse transcribed into cDNA using a high capacity-RNA-to-cDNA Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and PCR amplified using PrimeSTAR GXL DNA polymerase (Takara Bio) and a primer set. The PCR product was purified by a QIAquick PCR Purification Kit (Qiagen) and analyzed by High Sensitivity D1000 Screen Tape using an Agilent 4200 TapeStation (Agilent Technologies, Santa Clara, CA).
The total RNA in mouse TA or GC was extracted by Qiazol (Qiagen) and an RNeasy 96 Kit (Qiagen) according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed into cDNA using a high capacity-RNA-to-cDNA Kit according to the manufacturer’s instructions. Standard DNAs for skipped or unskipped products or 100 ng of cDNA products were PCR amplified by FastStart Universal Probe Master (Roche, Basel, Switzerland) and appropriate primer/probe sets (Table 1). The amount of each PCR product was calculated by using standard DNA, and the exon skipping efficiency was determined by the following formula: 100 × amount of skipped product/(amount of skipped product + amount of unskipped product).
Dystrophin protein detection by Western blotting
Harvested cells or collected tissues were mixed with RIPA buffer (FUJIFILM Wako Pure Chemical) containing protease inhibitor cocktail (Sigma-Aldrich) and EDTA (FUJIFILM Wako Pure Chemical) and homogenized. The homogenates were centrifuged, and the protein concentrations of the collected supernatants were determined with a Pierce 660 nm Protein Assay Kit (Thermo Fisher Scientific). Then, thermal denaturation was conducted with SDS sample buffer (Thermo Fisher Scientific or Bio-Rad) containing reducing agent (Thermo Fisher Scientific). The denatured samples were loaded onto a 3–8% tris-acetate gel (Thermo Fisher Scientific) for dystrophin detection or a TGX Any kD gel (Bio-Rad, Hercules, CA) for Cas9 and GAPDH detection. Proteins were transferred to PVDF membrane by the Trans-Blot Turbo system (Bio-Rad). Blotting was performed with polyclonal anti-dystrophin (2,000-fold dilution, cat #ab15277, Abcam, Cambridge, UK), monoclonal anti-GAPDH (4,000-fold dilution, cat #2118, Cell Signaling Technology, Danvers, MA) or polyclonal anti-Cas9 (1,000-fold dilution, cat #632607, Takara Bio) and secondary anti-rabbit IgG HRP-linked whole antibody (GE Healthcare, Chicago, IL) using iBind Western Systems (Thermo Fisher Scientific) according to the manufacturer’s instructions. Finally, the membranes were incubated in ECL Prime (GAPDH) or Select (dystrophin and Cas9) Western Blotting (GE Healthcare, Chicago, IL) and imaged on a ChemiDoc MP (Bio-Rad).
Measurement of genome editing efficiency
Genomic DNA was extracted from mouse skeletal muscle tissue (GC) by QIAamp Fast DNA Tissue Kit (Qiagen) according to the manufacturer’s instructions. For T7EI (T7 Endonuclease I) assay for mouse Rosa26 sgRNA cleavage activity, target region was amplified by PCR (forward primer; 5′-CTCCGAGGCGGATCACAAGCAATAATAACCTGTAG-3′, reverse primer; 5′-TGCAAGCACGTTTCCGACTTGAGTTGCCTCAAGAG-3′) using PrimeSTAR GXL DNA polymerase (Takara bio) and the PCR amplicons were purified using QIAquick PCR purification kit (Qiagen) according to the manufacturer’s instructions. Purified PCR amplicons were denatured and annealed in NEBuffer 2 (New England Biolabs, Ipswich, MA) and then digested with T7 endonuclease I (New England Biolabs) for 15 min at 37 °C. The resultant DNA fragments were analyzed by High Sensitivity D1000 Screen Tape using an Agilent 4200 TapeStation (Agilent Technologies).
For ddPCR (droplet digital PCR) analysis to assess the copy number loss at the hEx45 sgRNA #1 targeting region, genomic DNA was mixed with 2× ddPCR Supermix for Probes (no dUTP) (Bio-Rad) and two sets of primers/probes (Table 2) to amplify the sgRNA target and non-target sites. Then, droplets were generated using QX200 AutoDG (Bio-Rad) and subjected to PCR amplification and quantification using QX200 Droplet Digital PCR system (Bio-Rad).
Immunohistochemistry for detecting dystrophin and hematoxylin and eosin (H&E) staining
Fresh-frozen TA were sliced into 10 μm thick sections by a cryostat. The sections were reacted with 100-fold diluted polyclonal anti-dystrophin (cat #ab15277, Abcam, Cambridge, UK) or 1000-fold diluted polyclonal anti-Laminin 2 alpha (cat #ab11576, Abcam, Cambridge, UK) for 2 h at room temperature or overnight at 4 °C and then reacted with goat anti-Rabbit IgG (H;L) antibody conjugated with Alexa568 (cat #A11036, ThermoFisher, 200-fold dilution) for dystrophin detection or biotinylated IgG antibody (cat #BA-4000, Vector laboratories, 200-fold dilution) for laminin 2 alpha detection as the secondary antibody and ABC complex (Vector laboratories, CA) for 1–2 h at room temperature. The numbers of dystrophin fibers and central nuclei were counted by using HALO (Indica labs, Albuquerque, NM).
Sequence of dystrophin gene in hEx45KI-mdx44 by Sanger method
Reverse transcription products of GC in hEx45KI-mdx44 mice were PCR amplified with Q5 high-fidelity DNA polymerase 2× Master Mix (New England Biolabs) and forward (design of mouse Dmd exon 43) and reverse (design of mouse Dmd exon 46) primers and then purified with a QIAquick PCR Purification Kit according to the manufacturer’s instructions. Purified PCR products were amplified and purified with a BigDye terminatorv3.1 Cycle sequencing Kit according to the manufacturer’s instructions and sequenced with a 3500 Genetic analyzer by the Sanger method (Thermo Fisher Scientific).
Measurement of plasma creatine kinase (CK) as DMD biomarker
Blood was collected from the inferior vena cava and centrifuged at 20,400 × g for 5 min at 4 °C. CK in collected plasma was assayed by the enzymatic method.
Off-target analysis in vitro by the CIRCLE-seq method
For the CIRCLE-seq analysis36, genomic DNA was extracted from the human iPS cell line 1383D237. A Covaris E210 was used to shear the purified genomic DNA to an average length of 300 bp. The seared genomic DNA was circularized and digested with Cas9 protein (Axcelead) and in vitro transcribed sgRNA in digestion buffer containing 20 mM HEPES, 150 mM KCl, 1 mM DTT, 1 mM MgCl2, 50 µg/µl bovine serum albumin, and 10% glycerol. The digested DNA was processed to prepare the Illumina sequencing library by NEBNext Adaptor (cat #E7601A, NEB) ligation. The resultant libraries were quantified using a KAPA Library Quant Kit ABI Prism qPCR Mix for Illumina (KAPA biosystems) and sequenced with 75 bp paired-end reads on an Illumina NextSeq instrument.
Illumina sequencing adapter and low-quality reads (Quality score < 20) were removed from the resultant FASTQ files by cutadapt software (Version 2.4 with Python 3.4.10). Demultiplexed sequence reads were mapped on human genome hg38 with BWA-MEM (version: bwa-0.7.17-r1188). Among the mapped reads, CRISCLE-seq-specific paired-reads with opposite orientations were extracted using the 83, 163 or 99, 147 flags on SAM files, and the size of the deleted region was less than 20 bp. Filtered SAM files were converted to sorted BAM files by samtools (Version 0.1.19-44428 cd). By using bedtools (Version 2.27.1), the BAM files were converted to bedgraph files with peak height and location in the genome. Then, the peaks found in the negative control (Cas9/sgRNA untreated) were subtracted from the peaks obtained in the samples treated with each sgRNA by using bedtools with the intersect option. Finally, the common peaks between duplicated experiments were merged using bedtools intersect. The peak heights (log10) based on the chromosomal position were plotted using R (Version 3.6.1).
Cell-based off-target analysis
A piggyBac vector stably expressing sgRNA targeting human DMD exon 45 (#1) was constructed previously37. The sgRNA sequence against DMD exon 45 (#23) was inserted into the BamHI-EcoRI site of pPV-H1-ccdB-mEF1α-RiH plasmid (Addgene ID: 100598) to generate PB-H1-sgRNA-DMD23 vector. Then, we established a HEK293T cell line harboring a Dox- and dexamethasone (Dex)-inducible Cas9 CRONUS system37 by the transfection of pPV-TetO-SphcCas9-hGR-iC-EF1a-rtTA-iP (CRONUS-Puro, Addgene ID: 100596) and PB-H1-sgRNA (DMD #1 or DMD #23) with PBase using Lipofectamine 2000 (Thermo Fisher Scientific). Two days after the transfection, puromycin and hygromycin selection was applied for two weeks to select the cells with stably integrated Cas9-GR and sgRNA. The resultant cells were treated with 6 µM Dox to induce Cas9-GR expression and 1 µM Dex to induce the nuclear shuttling of Cas9-GR for 56 days, with passaging done every 3 days. Subsequently, genomic DNA was extracted and used for T7EI assays to assess mutations at the on-target and candidate off-target sites identified by the CIRCLE-seq analysis.
Statistical analysis
To compare two groups, we used two-sided Aspin–Welch’s t-test. To compare three groups, we used the two-sided Steel-Dwass test. P < 0.05 was considered statistically significant. Statistical analysis was conducted by EXSUS 2014 (Version 8.0, SAS 9.3 TS Levle1M2) from the SAS Institute, Inc. (NC).
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
